WO2014011819A2

WO2014011819A2 - Factor viii complex with xten and von willebrand factor protein, and uses thereof

Info

Publication number: WO2014011819A2
Application number: PCT/US2013/049989
Authority: WO
Inventors: Ekta Seth CHHABRA; Tongyao Liu; Pei-Yun Chang; Robert T. Peters; John KULMAN
Original assignee: Amunix Operating Inc.; Biogen Idec Ma Inc.
Priority date: 2012-07-11
Filing date: 2013-07-10
Publication date: 2014-01-16
Also published as: ES2770501T3; MX2015000397A; BR112015000267B1; CO7170123A2; CN104661674A; JP2022000471A; US20220056108A1; EP2882450B1; JP2015527882A; LT2882450T; JP2023166024A; EP2882450A4; PT2882450T; KR102329315B1; AU2013290173B2; KR102403545B1; US11091534B2; JP2018102323A; JP2020078352A; AU2018203206A1

Abstract

The present invention provides a chimeric protein comprising a VWF protein comprising the D' domain and D3 domain of VWF, one or more XTEN sequence, and a FVIII protein, wherein the VWF fragment, the XTEN sequence, or the FVIII protein are linked to or associated with each other. The chimeric protein can further comprise one or more Ig constant region or a portion thereof (e.g., an Fc region). A polypeptide chain comprising a VWF fragment of the invention binds to or is associated with a polypeptide chain comprising a FVIII protein linked to an XTEN sequence and the polypeptide chain comprising the VWF fragment can prevent or inhibit binding of endogenous VWF to the FVIII protein linked to the XTEN sequence. By preventing or inhibiting binding of endogenous VWF to the FVIII protein, which is a half-life limiting factor for FVIII, the VWF fragment can induce extension of half-life of the chimeric protein comprising a FVIII protein. The invention also includes nucleotides, vectors, host cells, methods of using the VWF fragment, or the chimeric proteins.

Description

FACTOR VIII COMPLEX WITH XTEN AND VON WILLEBRAND FACTOR

PROTEIN, AND USES THEREOF

BACKGROUND OF THE INVENTION

[0001] Haemophilia A is a bleeding disorder caused by defects in the gene

encoding coagulation factor VIII (FVIII) and affects 1-2 in 10,000 male births. Graw et al, Nat. Rev. Genet. 6(6): 488-501 (2005). Patients affected with hemophilia A can be treated with infusion of purified or recombinantly produced FVIII. All commercially available FVIII products, however, are known to have a half-life of about 8-12 hours, requiring frequent intravenous administration to the patients. See Weiner M.A. and Cairo, M.S., Pediatric Hematology Secrets, Lee, M.T., 12. Disorders of Coagulation, Elsevier Health Sciences, 2001; Lillicrap, D. Thromb. Res. 122 Suppl 4:S2-8 (2008). In addition, a number of approaches have been tried in order to extend the FVIII half-life. For example, the approaches in development to extend the half-life of clotting factors include pegylation, glycopegylation, and conjugation with albumin. See Dumont et al, Blood.

119(13): 3024-3030 (Published online Jan. 13, 2012). Regardless of the protein engineering used, however, the long acting FVIII products currently under development are reported to have limited half-lives - only to about 1.5 to 2 hours in preclinical animal models. See id. Consistent results have been demonstrated in humans, for example, rFVIIIFc was reported to improve half-life up to ~ 1.7 fold compared with AD V ATE® in hemophilia A patients. See Id. Therefore, the half-life increases, despite minor improvements, may indicate the presence of other Tl/2 limiting factors. See Liu, T. et al, 2007 ISTH meeting, abstract #P-M- 035; Henrik, A. et al, 2011 ISTH meeting, abstract #P=MO-181; Liu, T. et al, 2011 ISTH meeting abstract #P-WE-131.

[0002] Plasma von Willebrand Factor (VWF) has a half-life of approximately 12 hours (ranging from 9 to 15 hours).

http://www.nhlbi.nih.gov/guidelines/vwd/2_scientificoverview.htm (last visited October 22, 2011). The VWF half-life may be affected by a number of factors: glycosylation pattern, ADAMTS-13 (a disintegrin and metalloprotease with thrombospondin motif- 13), and various mutations in VWF.

[0003] In plasma, 95-98% of FVIII circulates in a tight non-covalent complex with full-length VWF. The formation of this complex is important for the maintenance of appropriate plasma levels of FVIII in vivo. Lenting et al., Blood. 92(11): 3983-96 (1998); Lenting et al, J. Thromb. Haemost. 5(7): 1353-60 (2007). The full-length wild-type FVIII is mostly present as a heterodimer having a heavy chain (MW 200kD) and a light chain (MW 73kD). When FVIII is activated due to proteolysis at positions 372 and 740 in the heavy chain and at position 1689 in the light chain, the VWF bound to FVIII is removed from the activated FVIII. The activated FVIII, together with activated factor IX, calcium, and phospholipid ("tenase complex"), induces the activation of factor X, generating large amounts of thrombin. Thrombin, in turn, then cleaves fibrinogen to form soluble fibrin monomers, which then spontaneously polymerize to form the soluble fibrin polymer. Thrombin also activates factor XIII, which, together with calcium, serves to crosslink and stabilize the soluble fibrin polymer, forming crosslinked (insoluble) fibrin. The activated FVIII is cleared fast from the circulation by proteolysis.

[0004] Due to the frequent dosing and inconvenience caused by the dosing

schedule, there is still a need to develop FVIII products requiring less frequent administration, i.e., a FVIII product that has a half-life longer than the 1.5 to 2 fold half-life limitation.

BRIEF SUMMARY OF THE INVENTION

[0005] The present invention is directed to a chimeric protein comprising (i) a von

Willebrand Factor (VWF) fragment comprising the D' domain and the D3 domain of VWF, (ii) an XTEN sequence, and (iii) a FVIII protein, wherein the VWF fragment and the XTEN sequence are linked by an optional linker and wherein the VWF fragment or the XTEN sequence is linked to or associated with the FVIII protein. The chimeric protein can comprise a single polypeptide chain comprising the VWF fragment, the XTEN sequence, and the FVIII protein, or two

polypeptide chains, a first chain comprising the VWF fragment and the second chain comprising the FVIII protein, wherein the XTEN polypeptide is linked either to the VWF fragment or the FVIII protein.

[0006] In one embodiment, the chimeric protein of the invention comprises a formula comprising:

(a) V-X-FVIII, (b) FVIII-X-V,

(c) V-X:FVIII,

(d) X-V:FVIII,

(e) FVIILV-X, or

(f) FVIILX-V,

wherein V comprises a VWF fragment,

X comprises one or more XTEN sequences, and

FVIII comprises a FVIII protein. The hyphen (-) can be a peptide bond or a linker, e.g., a cleavable linker, while the colon (:) represents a chemical association or a physical association between the polypeptides, for example a covalent or non-covalent bond.

[0007] In another embodiment, the chimeric protein further comprises (iv) an immunoglobulin (Ig) constant region or a portion thereof (also indicated as Fl or a first Ig constant region or a portion thereof) linked to the VWF fragment, the XTEN sequence, the FVIII protein, or any combinations thereof. In other embodiments, the chimeric protein further comprises an additional Ig constant region or a portion thereof (also indicated as F2 or a second Ig constant region or a portion thereof). The first Ig constant region or a portion thereof can be linked to the VWF fragment or the XTEN sequence, and the second Ig constant region can be linked to the FVIII protein. The first Ig constant region, the second Ig constant region or a portion thereof, or both can extend the half-life of the FVIII protein.

[0008] In some embodiments, the second Ig constant region or a portion thereof

(F2) is linked to the VWF fragment by a linker, e.g., a processable linker. In other embodiments, the second Ig constant region or a portion thereof (F2) is associated with the (first) Ig constant region or a portion thereof (Fl). The second Ig constant region or a portion thereof (F2) and the first Ig constant region or a portion thereof (Fl) can be identical or different. The second Ig constant region or a portion thereof can be associated with the Ig constant region or a portion thereof by a covalent bond, e.g., a disulfide bond. The VWF fragment linked to the first Ig constant region or a portion thereof may also be associated with the FVIII protein linked to the second Fc region by a non-covalent bond. In certain embodiments, the FVIII protein can further comprise one or more additional XTEN sequences which are linked to the C-terminus or N-terminus of the FVIII protein or inserted immediately downstream of one or more amino acids in the FVIII protein (e.g., one or more XTEN insertion sites). In some embodiments, the half-life of the FVIII protein is extended, compared to wild type FVIII or a FVIII protein without the VWF fragment.

[0009] In some embodiments, the chimeric protein comprises a formula

comprising:

(g) V-L2-X-L1-F1 :FVm-L3-F2;

GO V-L2-X-L1-F1 :F2-L3-FVIII;

(i) F 1 -L 1 -X-L2-V : FVIII-L3 -F2;

0⁾ F 1 -L 1 -X-L2-V:F2-L3 -FVIII;

(k) V-L2-X-L 1 -F 1 -L4-FVIII-L3 -F2;

(1) F2-L3-FVIII-L4-F 1 -L 1 -X-L2-V;

(m) FVIII-L3-F2-L4-V-L2-X-L1-F1; or

(n) F 1 -L 1 -X-L2-V-L4-F2-L3-FVIII,

wherein V comprises a VWF fragment,

each of LI, L2, and L3 comprises an optional linker, e.g., a cleavable linker,

L4 is an optional linker, e.g., a processable linker,

FVIII comprises a FVIII protein,

X comprises one or more XTEN sequences,

Fl comprises an optional first Ig constant region or a portion thereof,

F2 comprises an optional second Ig constant region or a portion thereof, and (:) is a covalent bond or non-covalent bond.

[0010] The present invention is also directed to a chimeric protein comprising (i) a

FVIII protein, (ii) an XTEN sequence, and (iii) an Ig constant region or a portion thereof, wherein the XTEN sequence is linked to the FVIII protein by an optional linker at the N-terminus or C terminus of the FVIII protein or inserted

immediately downstream of one or more amino acids in the FVIII protein (e.g., one or more insertion sites) and wherein the Ig constant region or a portion thereof is linked to or associated with the FVIII protein or the XTEN sequence. In one embodiment, the Ig constant region or a portion thereof useful for the chimeric protein comprises a first Fc region. In another embodiment, the chimeric protein further comprises an additional Ig constant region or a portion thereof. The additional Ig constant region or a portion thereof useful for the invention can comprise a second Fc region, which is linked to or associated with the first Fc region, e.g., by a covalent bond. In other embodiments, the first Fc region is linked to the second Fc region by a linker, e.g., a processable linker.

In other aspects, a chimeric protein comprises (i) a FVIII protein, (ii) an

XTEN sequence, (iii) a VWF fragment, and (iv) an Ig constant region or a portion thereof, which comprises the D' domain and the D3 domain of VWF, wherein the XTEN sequence is linked to the FVIII protein by an optional linker at the N- terminus or C terminus of the FVIII protein or inserted immediately downstream of one or more amino acids in the FVIII protein {e.g., one or more insertion sites), the VWF fragment is linked to or associated with the FVIII protein or the XTEN sequence, and the Ig constant region or a portion thereof is linked to the FVIII protein, the XTEN sequence, the VWF fragment, or any combinations thereof. Non-limiting examples of the chimeric proteins may comprise a formula, which comprises:

(1) FVIII(X1 )-L 1 -F 1 : V-L2-X2-L3-F2;

(2) FVIII(X1 )-L 1 -F 1 :F2-L3-X2-L2-V;

(3) F 1 -L 1 -FVIII(X 1 ) : V-L2-X2-L3 -F2;

(4) F 1 -L 1 -FVIII(X 1 ) :F2-L3 -X2-L2-V;

(5) FVIII(X1 )-L 1 -F 1 -L4-V-L2-X2-L3-F2;

(6) FVIII(X1 )-L 1 -F 1 -L4-F2-L3-X2-L2-V;

(?) F 1 -L 1 -FVIII(X1 )-L4-V-L2-X2-L3-F2, or

(8) F 1 -L 1 -FVIII(X1 )-L4-F2-L3-X2-L2-V,

wherein FVIII(Xl) comprises a FVIII protein and one or more XTEN sequences, wherein one or more of the XTEN sequences are linked to the N-terminus or C- terminus of the FVIII protein or inserted immediately downstream of one or more amino acids in the FVIII protein {e.g., one or more XTEN insertion sites);

each of LI, L2, or L3 comprises an optional linker, e.g., a cleavable linker;

L4 is a linker, a processable linker;

X2 comprises one or more XTEN sequences;

Fl comprises an Ig constant region or a portion thereof;

F2 comprises an optional additional Ig constant region or a portion thereof, and

V comprises a VWF fragment;

(-) is a peptide bond or one or more amino acids; and (:) comprises a covalent bond or a non-covalent bond.

[0012] One aspect of the invention is that the VWF fragment useful for the

chimeric protein does not bind to a VWF clearance receptor, which prevents or inhibits interaction of the FVIII protein with endogenous VWF. The chimeric protein comprising the VWF fragment thus has reduced clearance or is not cleared through a VWF clearance pathway. Another aspect of the invention is that the VWF fragment is capable of protecting the FVIII protein from one or more protease cleavages, protecting the FVIII protein from activation, stabilizing the heavy chain and/or the light chain of the FVIII protein, or preventing clearance of the FVIII protein by one or more scavenger receptors.

[0013] Because of the VWF fragment's ability to prevent or inhibit interaction between the FVIII protein and endogenous VWF, the half-life of the FVIII protein, is extended compared to a FVIII protein without the VWF fragment. In one embodiment, the half-life of the FVIII protein is extended at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, or at least about 12 times longer than wild type FVIII. In another embodiment, the half-life of the FVIII protein is at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours.

[0014] The Ig constant region or a portion thereof useful for the chimeric protein comprises a first Fc region, which is linked to the VWF fragment by an optional linker, e.g., a cleavable linker. The chimeric protein can further comprise an additional Ig constant region or a portion thereof, which is linked to the FVIII protein or the XTEN sequence, the Ig constant region or a portion thereof, the VWF fragment, or any combinations thereof by an optional linker. In one embodiment, the additional Ig constant region or a portion thereof is linked to the FVIII protein by an optional linker. The additional Ig constant region or a portion thereof can comprise a second Fc region.

[0015] The Ig constant region or a portion thereof useful in the present invention and the additional Ig constant region or a portion thereof useful in the present invention are identical or different.

[0016] In some aspects, the FVIII protein is linked to an XTEN sequence at the C- terminus or the N-terminus of the FVIII protein or inserted immediately downstream of one or more amino acids in mature native human FVIII (e.g., one or more insertion sites) or any combinations thereof. One or more insertion sites in the FVIII protein can be located within one or more domains of the FVIII protein selected from the group consisting of the Al domain, the al acidic region, the A2 domain, the a2 acidic region, the A3 domain, the B domain, the CI domain, the C2 domain, and any combinations thereof or between one or more domains of the FVIII protein selected from the group consisting of the Al domain and al acidic region, the al acidic region and A2 domain, the A2 domain and a2 acidic region, the a2 acidic region and B domain, the B domain and A3 domain, the A3 domain and CI domain, the CI domain and C2 domain, and any combinations thereof or between two domains of the FVIII protein selected from the group consisting of the Al domain and al acidic region, the al acidic region and A2 domain, the A2 domain and a2 acidic region, the a2 acidic region and B domain, the B domain and A3 domain, the A3 domain and C 1 domain, the C 1 domain and C2 domain, and any combinations thereof.

[0017] In one embodiment, the one or more insertion sites are located

immediately downstream of one or more amino acids in mature native human FVIII (e.g., SEQ ID NO: 4 [mature FVIII sequence-full length]) selected from the group consisting of the amino acid residues in Table 7, 8, 9, 10, 1 1 , or any combinations thereof.

[0018] In another embodiment, the one or more insertion sites are located in one or more permissive loops of mature native human FVIII. In other embodiments, the one or more insertion sites are located in the a3 region of mature native human FVIII. For example, an XTEN sequence can be inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO: 4 (full length mature FVIII). In other embodiments, a FVIII protein is linked to at least two XTEN sequences, a first XTEN sequence inserted within the a3 region, and a second XTEN sequence inserted within a permissive loop in the FVIII protein (e.g., Al-1 , Al-2, A2-1 , A2- 2, A3-1 , or A3-2). In still other embodiments, a FVIII protein is linked to at least three XTEN sequences, a first XTEN sequence inserted within the a3 region and a second XTEN sequence and a third XTEN sequence inserted within one or two permissive loop in the FVIII protein (e.g., Al-1 , Al-2, A2-1 , A2-2, A3-1 , or A3- 2)·

In certain embodiments, the one or more insertion sites for one or more

XTEN insertions are immediately downstream of one or more amino acids (numbered relative to mature FVIII sequence) selected from the group consisting of:

(1) amino acid 3, (2) amino acid 18, (3) amino acid 22,

(4) amino acid 26, (5) amino acid 32, (6) amino acid 40,

(7) amino acid 60, (8) amino acid 65, (9) amino acid 81 ,

(10) amino acid 1 16, (1 1) amino acid 1 19, (12) amino acid 130,

(13) amino acid 188, (14) amino acid 21 1 , (15) amino acid 216,

(16) amino acid 220, (17) amino acid 224, (18) amino acid 230,

(19) amino acid 333, (20) amino acid 336, (21) amino acid 339,

(22) amino acid 375, (23) amino acid 399, (24) amino acid 403,

(25) amino acid 409, (26) amino acid 416, (26) amino acid 442,

(28) amino acid 487, (29) amino acid 490, (30) amino acid 494,

(31) amino acid 500, (32) amino acid 518, (33) amino acid 599,

(34) amino acid 603, (35) amino acid 713, (36) amino acid 745,

(37) amino acid 1656, (38) amino acid 171 1 , (39) amino acid 1720,

(40) amino acid 1725, (41) amino acid 1749, (42) amino acid 1796,

(43) amino acid 1802, (44) amino acid 1827, (45) amino acid 1861 ,

(46) amino acid 1896, (47) amino acid 1900, (48) amino acid 1904,

(49) amino acid 1905, (50) amino acid 1910, (51) amino acid 1937, (52) amino acid 2019, (53) amino acid 2068, (54) amino acid 2111,

(55) amino acid 2120, (56) amino acid 2171, (57) amino acid 2188,

(58) amino acid 2227, (59) amino acid 2277, and

(60) two or more combinations thereof.

[0020] In some embodiments, one XTEN is inserted in the FVIII protein. In some embodiments, two XTENs are inserted in the FVIII protein. In some

embodiments, 3 XTENs are inserted in the FVIII protein.

[0021] In a particular example, a first XTEN is inserted immediately downstream of amino acid 26 corresponding to SEQ ID NO: 4, and a second XTEN is inserted immediately downstream of amino acid 1720 corresponding to SEQ ID NO: 4 (full-length mature FVIII). In another example, a first XTEN is inserted immediately downstream of amino acid 403 corresponding to SEQ ID NO: 4, and a second XTEN is inserted immediately downstream of amino acid 1720 corresponding to SEQ ID NO: 4. In some examples, a first XTEN is inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO: 4, and a second XTEN is inserted immediately downstream of amino acid 1720 corresponding to SEQ ID NO: 4. In other examples, a first XTEN is inserted immediately downstream of amino acid 26 corresponding to SEQ ID NO: 4, a second XTEN is inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO: 4, and a third XTEN is inserted immediately downstream of amino acid 1720 corresponding to SEQ ID NO: 4. In yet other embodiments, a first XTEN is inserted immediately downstream of amino acid 403 corresponding to SEQ ID NO: 4, a second XTEN is inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO: 4, and a third XTEN is inserted immediately downstream of amino acid 1720 corresponding to SEQ ID NO: 4. In still other embodiments, a first XTEN is inserted between amino acids 403 and 404 corresponding to SEQ ID NO: 4, a second XTEN is inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO: 4, and a third XTEN is inserted immediately downstream of amino acid 1720 corresponding to SEQ ID NO: 4. In certain embodiments, a first XTEN is inserted immediately downstream of amino acid 26 corresponding to SEQ ID NO: 4 (full- length mature FVIII), a second XTEN is inserted immediately downstream of amino acid 1720 corresponding to SEQ ID NO: 4, and a third XTEN is inserted immediately downstream of amino acid 1900 corresponding to SEQ ID NO: 4. In some embodiments, a first XTEN is inserted immediately downstream of amino acid 26 corresponding to SEQ ID NO: 4, a second XTEN is inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO: 2, a third XTEN is inserted immediately downstream of amino acid 1720 corresponding to SEQ ID NO: 4, and a fourth XTEN is inserted immediately downstream of amino acid 1900 corresponding to SEQ ID NO: 4. In another example, an XTEN is inserted immediately downstream of amino acid 745 corresponding to SEQ ID NO: 4. In an additional example, a first XTEN is inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO: 4 and a second XTEN is inserted immediately downstream of amino acid 1900 corresponding to SEQ ID NO: 4. In some embodiments, a first XTEN is inserted immediately downstream of amino acid 26 corresponding to SEQ ID NO: 4, a second XTEN is inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO: 4, and a third XTEN is inserted immediately downstream of amino acid 1900 corresponding to SEQ ID NO: 4. In another example, a first XTEN is immediately inserted downstream of amino acid 403 corresponding to SEQ ID NO: 4 and a second XTEN is inserted immediately downstream of amino acid 745 corresponding to SEQ ID NO: 4. In some embodiments, a first XTEN is inserted immediately downstream of amino acid 745 of corresponding to SEQ ID NO: 4, and a second XTEN is inserted immediately downstream of amino acid 1900 corresponding to SEQ ID NO: 4. In some embodiments, a first XTEN is inserted immediately downstream of amino acid 18 corresponding to SEQ ID NO: 4, and a second XTEN is inserted immediately downstream of amino acid 745 corresponding to SEQ ID NO: 4.

[0022] In some embodiments, the FVIII protein is a dual chain FVIII isoform. In some embodiments, the FVIII protein is a single chain FVIII isoform.

[0023] In some embodiments, the XTEN that is inserted is SEQ ID NO: 39

(AE288). In some examples, the XTENs that are inserted are SEQ ID NOs: 38 and 37 (AG144 and AE144). In some examples, the XTENs that are inserted are SEQ ID NOs: 37, 38 and 37 (AE144, AG144, and AE144). In some embodiments, the XTENs that are inserted are SEQ ID NOs: 37 and 40 (AE144 and AE288). In some embodiments, the XTENs that are inserted are AE42 (SEQ ID NO: 36), AE72 (SEQ ID NO: 127), AE144 2A (SEQ IDNO: 128), AE144 3B (SEQ ID NO: 129), AE144 4A (SEQ ID NO: 130), AE144 5A (SEQ IDNO: 131), AE144 6B (SEQ IDNO: 132), AG144 A (SEQ ID NO: 133), AG144 B (SEQ IDNO: 134), AG144 C (SEQ ID NO: 135), AG144 F (SEQ IDNO: 136), AE864 (SEQ ID NO: 43), AE576 (SEQ ID NO: 41), AE288 (SEQ IDNO: 39), AE288 2 (SEQ ID NO: 137), AE144 (SEQ ID NO: 37), AG864 (SEQ ID NO: 44), AG576 (SEQ ID NO: 42), AG288 (SEQ ID NO: 40), AG144 (SEQ ID NO: 38), and any combinations thereof.

[0024] The FVIII protein useful in the invention can comprise B domain or a portion thereof, e.g., SQ B domain deleted FVIII. In one embodiment, the FVIII protein comprises single chain FVIII. In another embodiment, the single chain FVIII contains at least one amino acid substitution at a residue corresponding to residue 1648, residue 1645, or both of full-length mature Factor VIII polypeptide (SEQ ID NO: 4) or residue 754, residue 751 , or both of SQ BDD Factor VIII (SEQ ID NO: 6). In other embodiments, the amino acid substitution is an amino acid other than arginine. In some embodiments, the FVIII protein comprises a heavy chain of FVIII and a light chain of FVIII, wherein the heavy chain and the light chain are associated with each other by a metal bond.

[0025] The FVIII protein can have a low affinity to or does not bind to a low- density lipoprotein receptor-related protein (LRP), e.g., by containing at least one amino acid substitution that lowers the affinity to or eliminates the binding to the LRP. Such at least one amino acid substitution can be at a residue corresponding to residue 471, residue 484, residue 487, residue 490, residue 497, residue 2092, residue 2093 or two or more combinations thereof of full-length mature FVIII. In a particular embodiment, the amino acid substitution at residue 471, 484, or 497 is an amino acid other than arginine, the amino acid substitution at residue 487 is an amino acid other than tyrosine, the amino acid substitution at residue 2092 is an amino acid other than lysine, or the amino acid substitution at residue 2093 is an amino acid other than phenylalanine.

[0026] In some embodiments, the FVIII protein contains at least one amino acid substitution, which induces the FVIII protein to be more stable than a FVIII protein without the substitution. Such substitutions can be located in the A2 domain and the A3 domain of the FVIII protein, e.g., at a residue corresponding to residue 664, residue 1826, residue 662, residue 1828, or two or more combinations thereof of full-length mature FVIII.

[0027] The VWF fragment useful for the present invention comprises a D' domain and D3 domain, which together are capable of binding to FVIII. The VWF fragment can comprise the amino acid sequence of the D' domain is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764 to 866 of SEQ ID NO: 2 and/or the amino acid sequence of the D3 domain is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 867 to 1240 of SEQ ID NO: 2. In one embodiment, the VWF fragment is a monomer. In another embodiment, the VWF fragment comprises at least two VWF fragments, at least three VWF fragments, at least four VWF fragments, at least five VWF fragments, or at least six VWF fragments. In one embodiment, the two or more VWF fragments may be identical or they may be different. The VWF fragment can comprise an amino acid at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764 to 1240 of SEQ ID NO: 2. The VWF fragment may consist essentially of or consist of amino acids 764 to 1240 of SEQ ID NO: 2. In certain embodiments, the VWF fragment can contain at least one amino acid substitution at a residue corresponding to residue 1099, residue 1142, or both residues 1099 and 1142 of SEQ ID NO: 2. In other embodiments, the VWF fragment further comprises the Dl domain, the D2 domain, or the Dl and D2 domains of VWF.

[0028] The VWF fragment may further comprise a VWF domain selected from the group consisting of the Al domain, the A2 domain, the A3 domain, the D4 domain, the Bl domain, the B2 domain, the B3 domain, the CI domain, the C2 domain, the CK domain, one or more fragments thereof, and any combinations thereof. For example, the VWF fragment can consist essentially of or consist of: (1) the D' and D3 domains of VWF or fragments thereof; (2) the Dl, D', and D3 domains of VWF or fragments thereof; (3) the D2, D', and D3 domains of VWF or fragments thereof; (4) the Dl, D2, D', and D3 domains of VWF or fragments thereof; or (5) the Dl, D2, D', D3, and Al domains of VWF or fragments thereof. In some embodiments, the VWF fragment further comprises a signal peptide of VWF or FVIII which is operably linked to the VWF fragment.

One or more of the linkers useful in the invention have a length of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, or 2000 amino acids. In some embodiments, one or more of the linkers have a length of about 1 to about 2000 amino acids. In one embodiment, one or more of the linkers have a length of at least about 20, 35, 42, 48, 73, 75, 95, 98, 144, 288, 324, 333, 576, or 864 amino acids. In another embodiment, one or more of the linkers comprise a gly/ser peptide, an XTEN sequence, or both. Examples of the gly/ser peptide include, but are not limited to, a formula of (Gly₄Ser)_n (SEQ ID NO: 139) or S(Gly₄Ser)_n (SEQ ID NO: 140), wherein n is a positive integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. For example, the (Gly₄Ser)_n linker can be (Gly₄Ser)₃ (SEQ ID NO: 63) or (Gly₄Ser)₄ (SEQ ID NO: 138). In one embodiment, the linker comprises at least one first cleavage site at the N-terminus of the linker, at least one second cleavage site at the C-terminus of the linker, or both. In another embodiment, the linker comprises 20 amino acids, 35 amino acids, 48 amino acids, 73 amino acids, or 95 amino acids thrombin cleavable linker. The cleavable linkers can comprise one or more of the cleavage sites by a protease selected from the group consisting of factor XIa, factor Xlla, kallikrein, factor Vila, factor IXa, factor Xa, factor Ila (thrombin), Elastase-2, Granzyme-B, TEV, Enterokinase, Protease 3C, Sortase A, MMP-12, MMP-13, MMP-17, and MMP-20, e.g., TLDPPvSFLLPvNPNDKYEPFWEDEEK (SEQ ID NO: 8). Non- limiting examples of one or more of the cleavage sites comprise an amino acid sequence selected from the group consisting of RRRR (SEQ ID NO: 9), RKRRKR (SEQ ID NO: 10), RRRRS (SEQ ID NO: 11), TQSFNDFTR (SEQ ID NO: 12), SVSQTSKLTR (SEQ ID NO: 13), DFLAEGGGVR (SEQ ID NO: 14), TTKIKPR (SEQ ID NO: 15), LVPRG (SEQ ID NO: 16), ALRPR (SEQ ID NO: 17), KLTRAET (SEQ ID NO: 18), DFTRVVG (SEQ ID NO: 19), TMTRIVGG (SEQ ID NO: 20), SPFRSTGG (SEQ ID NO: 21), LQVRIVGG (SEQ ID NO: 22), PLGRIVGG (SEQ ID NO:23), IEGRTVGG (SEQ ID NO: 24), LTPRSLLV (SEQ ID NO: 25), LGPVSGVP (SEQ ID NO: 26), VAGDSLEE (SEQ ID NO: 27), GPAGLGGA (SEQ ID NO: 28), GPAGLRGA (SEQ ID NO: 29), APLGLRLR (SEQ ID NO: 30), PALPLVAQ (SEQ ID NO: 31), ENLYFQG (SEQ ID NO: 32), DDDKIVGG (SEQ ID NO: 33), LEVLFQGP (SEQ ID NO: 34), and LPKTGSES (SEQ ID NO: 35). In some embodiments, the first cleavage site and the second cleavage site are identical or different.

[0030] The XTEN sequence useful for the invention can be selected from the group consisting of AE42 (SEQ ID NO: 36), AE144 (SEQ ID NO: 37), AG144 (SEQ ID NO: 38), AE288 (SEQ ID NO: 39), AG288 (SEQ ID NO: 40), AE576 (SEQ ID NO: 41). AG576 (SEQ ID NO: 42), AE864 (SEQ ID NO: 43), AE72 (SEQ ID NO: 127), AE144 2A (SEQ ID NO: 128), AE144 3B (SEQ ID NO: 129), AE144 4A (SEQ ID NO: 130), AE144 5A (SEQ ID NO: 131), AE144 6B (SEQ ID NO: 132), AG144 A (SEQ ID NO: 133), AG144 B (SEQ ID NO: 134), AG144 C (SEQ ID NO: 135), AG144 F (SEQ ID NO: 136), AE288 2 (SEQ ID NO: 137), or AG864 (SEQ ID NO: 44). In a particular embodiment, the XTEN sequence comprises AE288 or AG288.

[0031] The chimeric protein of the invention can be polysialylated, pegylated, or hesylated.

[0032] The present invention is also directed to a polynucleotide or a set of

polynucleotides encoding the chimeric protein. The polynucleotide can further comprise a polynucleotide chain, which encodes PC5 or PC7. The invention is also directed to a vector comprising the polynucleotide or the set of

polynucleotides and one or more promoter operably linked to the polynucleotide or the set of polynucleotides. The vector can further comprise an additional vector, which comprises a polynucleotide chain encoding PC5 or PC7. The invention is also drawn to a host cell comprising the polynucleotide or the vector. The host cell can be a mammalian cell, e.g., HEK293 cell, CHO cell, or BHK cell. In some embodiments, the PC5 or PC7 of the host cell cleaves the D1D2 domains ofVWF.

[0033] The invention is also directed to a pharmaceutical composition comprising the chimeric protein, the polynucleotide, the vector, or the host cell, and a pharmaceutically acceptable carrier. The composition of the invention thus has an extended half-life compared to wild type FVIII protein. The half-life of the FVIII protein is extended at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, or at least about 12 times longer than wild type FVIII. The half-life of Factor VIII is at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, at least about 28 hours, at least about 29 hours, at least about 30 hours, at least about 31 hours, at least about 32 hours, at least about 33 hours, at least about 34 hours, at least about 35 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours.

[0034] The composition of the present invention can be administered by a route selected from the group consisting of topical administration, intraocular administration, parenteral administration, intrathecal administration, subdural administration and oral administration. In one embodiment, the composition is administered via parenteral administration, e.g., intravenous or subcutaneous administration. The composition of the invention is useful to treat a bleeding disease or condition in a subject in need thereof. The bleeding disease or condition is selected from the group consisting of a bleeding coagulation disorder, hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, bleeding in the illiopsoas sheath and any combinations thereof. In one embodiment, the subject treated with the chimeric protein is scheduled to undergo a surgery. In another embodiment, the treatment is prophylactic or on-demand.

[0035] The invention is also directed to a method of preventing or inhibiting

binding of a FVIII protein with endogenous VWF comprising adding an effective amount of the chimeric protein, the polynucleotide vector, the host cell, or the composition to a subject in need thereof, wherein the VWF fragment binds to the FVIII protein and thus prevents or inhibits binding of endogenous VWF. The present invention is further directed to a method of extending or increasing the half-life of the FVIII protein, wherein the method comprises administering an effective amount of the chimeric protein, the polynucleotide, the vector, the host cell, or the composition to a subject in need thereof, wherein the VWF fragment binds to the FVIII protein and thus extends or increases the half-life of the FVIII protein. Also provided is a method of preventing or inhibiting clearance of a FVIII protein from a cell, wherein the method comprises administering an effective amount of the chimeric protein, the polynucleotide, the vector, the host cell, or the composition to a cell comprising a FVIII protein or a polynucleotide encoding the FVIII protein, wherein the protein having VWF activity binds to the FVIII protein. The subject useful for the present methods is an animal, e.g., a human, e.g., a patient suffering from hemophilia A.

[0036] The present invention also provides a method of treating a bleeding disease or disorder in a subject in need thereof comprising administering an effective amount of the chimeric protein, the polynucleotide, the vector, the host cell, or the composition, wherein the bleeding disease or disorder is selected from the group consisting of a bleeding coagulation disorder, hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, and bleeding in the illiopsoas sheath. The treatment can be prophylactic or on-demand. In one embodiment, the effective amount is 0.1 μg/kg to 500 mg/kg.

[0037] The invention also includes a method of making a chimeric protein,

comprising transfecting one or more host cell with the polynucleotide or the vector and expressing the chimeric protein in the host cell.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

[0038] Figure 1A-D. Schematic diagrams of VWF fragments. Fig. 1 A shows three exemplary VWF fragments useful for the invention, e.g., VWF-002, VWF- 010, and VWF-013. VWF-002 contains amino acids 1 to 477 of SEQ ID NO: 124 (amino acids 764 to 1240 of SEQ ID NO: 2) and is synthesized without the pre/propeptide sequences. VWF-010 contains the D1D2 domains in addition to the D'D3 domains. VWF-013 contains the D1D2DO3 domains in addition to alanine residues substituting cysteines at residues 336 and 379 of SEQ ID NO: 123 Fig. IB shows VWF-031, which contains the D1D2DO3 domains fused to an Ig constant region or a portion thereof, e.g., an Fc region, by a cleavable linker, e.g., a 48 amino acids thrombin cleavable linker. Fig. 1C shows VWF-025, which is a nucleotide sequence encoding D1D2DO3 domains contained in pLIVE vector, and VWF-029, which is a nucleotide sequence encoding D1D2DO3 domains with two amino acid substitutions, C336A and C379A, in pLIVE vector. Fig. ID shows full-length VWF fragment comprising propeptide (the Dl and D2 domains) and mature subunits (the D', D3, Al, A2, A3, D4, Bl-3, Cl-2 domains). The VWF fragment is about 250 kDa protein and forms multimers (> 20 MDa) by disulfide bonding. The VWF fragment associates with FVIII (95-98%) in non- covalent complex and then extends half-life of FVIII by protecting FVIII from protease cleavage/activation, stabilizing heavy & light chain, and preventing clearance of FVIII by scavenger receptors. The VWF fragment also can limit half-life of FVIII by clearance of FVIII- VWF complex through VWF receptors and preventing pinocytosis and recycling of rFVIIIFc.

[0039] Figure 2. Pharmacokinetic profile of rFVIII-XTEN (rFVIII-AE288 or rFVIII-288AE) in VWF D^*D3 expression mice or in FVIII and VWF double knockout (DKO) mice. Figure 2A shows the timeline of hydrodynamic injection (HDI) of the D^*D3 domain encoding plasmid DNA (VWF-025) (day -5), intravenous dosing of rFVIII-XTEN AE288 (day 0), and PK sample collection (day 5). Figure 2B shows FVIII activity measured by a FVIII chromogenic assay after IV dosing of rFVIII-XTEN288 in D1D2DO3 mice (inverted triangle) and rFVIII-XTEN288 in DKO mice (diamond). Fig. 2C shows the D^*D3 plasma level (ng/mL) after administration of VWF-025. The X axis represents time in hours.

[0040] Figure 3. Schematic diagram of exemplary VWF:FVIII heterodimer

constructs. The constructs have the common structure represented as formula FVIII-F1-L1-V-X-L2-F2, but contain examples of different variable linkers. The construct (FVIII- 161) shown contains a heterodimeric FVIII (the heavy chain and the light chain are associated by a metal bond) linked to a first Fc region and a VWF fragment, which is the D' and D3 domains of VWF {i.e., amino acids 1 to 477 of SEQ ID NO: 2 with amino acid substitutions C336A and C379A) linked to an XTEN sequence, which is further linked to a cleavable linker and a second Fc region. The XTEN sequence contained in FVIII-161 is an XTEN AE288 sequence, and the linker is a thrombin cleavable linker, which has 35 amino acids. In FVIII-161, the FVIII protein linked to the first Fc region is linked to the VWF fragment by a processable linker. Upon expression, the processable linker can be cleaved by an intracellular processing enzyme, thus making the construct three polypeptide chains associated with each other.

[0041] Figure 4 is schematic diagrams of FVIII- VWF heterodimer or monomer examples. FVIII-168, FVIII- 175, FVIII-172, FVIII-174, and FVIII170. Construct FVIII- 168 comprises a single chain FVIII sequence (having an alanine residue substitute the arginine residues at residues 1645 and 1648) linked to a first Fc region, which is then fused to a VWF fragment linked to a second Fc region by a thrombin cleavable linker, which has 48 amino acids. AE288 XTEN is inserted in the B domain of the single chain FVIII sequence. The linkage between the first Fc region and the VWF fragment comprises a linker that is capable of being cleaved by an intracellular processing enzyme, i.e., processable linker. Construct FVIII - 175 comprises a single chain FVIII (having an alanine residue substitute the arginine residues at residues 1645 and 1648) linked to AE288 XTEN and a first Fc region, which is linked to a second Fc region by a linker, e.g., a processable linker. AE288 XTEN is inserted in the B domain of the single chain FVIII sequence. Construct FVIII-172 comprises two polypeptide chains, a first chain comprising a heavy chain FVIII sequence fused to AE288 XTEN, a second chain comprising a light chain FVIII sequence, a first Fc region, a linker {e.g., a processable linker), a VWF fragment, a thrombin cleavable linker {e.g., 48 amino acids), and a second Fc region. Construct FVIII-174 comprises two polypeptide chains, a first chain comprising a heavy chain FVIII sequence fused to AE288 XTEN and a second chain comprises a light chain FVIII, a first Fc region, a linker {e.g., a processable linker), and a second Fc region. Construct FVIII- 170 comprises a VWF fragment, AE288 XTEN, a linker {e.g., a thrombin cleavable linker, which is 35 amino acids in length), and a single chain FVIII sequence.

[0042] Figure 5. Pharmacokinetic profile of FVIII/VWF heterodimers containing an XTEN sequence in combination with an Fc region. Constructs FVIII-161, FVIII-168, and FVIII-172 were administered to FVIII: VWF double knockout (DKO) mice by Hydrodynamic injection (HDI) at lOOug/mouse dose. Construct FVIII- 170 was administered to FVIII: VWF DKO mice by HDI at 50

dose. The post-HDI plasma FVIII activity was analyzed by FVIII chromogenic assay for 24 hr post-HDI. The FVIII activity of the FVIII: VWF heterodimers containing an XTEN sequence and Fc domains was compared with the FVIII activity of BDD-FVIII without the VWF fragment, XTEN sequence, and Fc domains.

[0043] Figure 6. Schematic diagrams of FVIII- VWF heterodimer examples co- transfection system. Fig. 6A. Construct FVIII- 169 contains the full-length FVIII sequence (with an alanine residue substituting the arginine residues at 1645 and 1648 and with an XTEN sequence inserted in the single chain FVIII sequence), which is linked to an Fc region. VWF-031 contains the D1D2D'D3 fragment (with an alanine residue substituting the cysteine residues at 336 and 379) which is linked to another Fc region with a 48 thrombin cleavable linker. After intracellular processing, construct FVIII- 169 produces a full length single chain FVIII (SCFVIII) fused to one Fc fragment and an XTEN sequence, and construct VWF-031 produces a 477 amino acid D'D3 fragment linked to another Fc fragment. Two covalent bonds can be formed between the Fc fragments that are linked to the SC FVIII or the D'D3 fragment, this in turn allows a non-covalent association of FVIII and D'D3. Fig. 6B. Construct FVIII- 173 contains a heterodimeric FVIII sequence, a heavy chain FVIII sequence linked to an XTEN sequence and a light chain FVIII sequence linked to an Fc region. VWF-031 is described above. After intracellular processing, construct FVIII- 173 produces a heterodimeric protein, a heavy chain FVIII fused to an XTEN sequence, a light chain FVIII fused to one Fc fragment, and construct VWF-031 produces a 477 amino acid D'D3 fragment linked to another Fc fragment. Two covalent bonds can be formed between the Fc fragments that are linked to the light chain FVIII or the D'D3 fragment, this in turn allows a non-covalent association of FVIII and D^*D3.

[0044] Figure 7. Binding Affinity of Exemplary FVIII: VWF containing an XTEN sequence and Fc domains to immobilized hVWF in Octet assay. The binding affinity for FVIII- 169/VWF-031 and FVIII-057 (rFVIIIFc) fused to immobilized hVWF was tested using biolayer interferometry based measurements (Octet assay). Figure 7 A shows binding response in nanomoles of FVIII 169 and FVIIIFc drug substance (a positive control) to immobilized hVWF. Figure 7B shows binding response of human IgGl (a negative control) to immobilized human VWF.

[0045] Figure 8. Pharmacokinetic (PK) profile of FVIII- 169 in HernA and

FVIILVWF double knockout (DKO) mice. Figure 8A shows the PK profile of FVIII- 169/VWF-031 and FVIIIFc in HemA mice. HemA mice were treated with a single intravenous dose of FVIII- 169/VWF-031 at 200 IU/kg. Plasma samples collected from the mice were tested by FVIII chromogenic assay. Half-life of FVIII- 169/VWF-031 was calculated using WinNonlin program. Figure 8B shows the PK profile of FVIII- 169/VWF-031, FVIII- 169/Fc, and FVIIIFc in FVIII/VWF DKO mice.

[0046] Figure 9. PK profile of FVIII-XTEN variants in D'D3 expressing

FVIII/VWF DKO mice. Figure 9A shows comparison of the PK profile of the FVIII-XTEN variants, FVIII with one XTEN, FVIII with two XTENs, and FVIII with three XTENs. One, two, or three XTENs were inserted in various portions of FVIII including C-terminus and B-domain. CT indicates that an XTEN is linked to the C-terminus of FVIII. Insertion site B/CT indicates that one XTEN is inserted between amino acid residue 745 and amino acid residue 746 of the FVIII protein and another XTEN is linked to the C-terminus of the FVIII protein. The amino acid residue numbering corresponds to the SQ BDD FVIII protein sequence. Insertion site 1900/B/CT indicates that a first XTEN is inserted between amino acid residue 1900 and amino acid residue 1901 of FVIII, a second XTEN is inserted between amino acid residue 745 and amino acid residue 746 of FVIII, and a third XTEN is linked to the C-terminus of FVIII. The mouse strain used to administer the FVIII-XTEN variants is a DKO mouse strain expressing D'D3 domains. Figure 9B shows the PK profile of FVIII-XTEN with three XTEN insertions. The FVIII-XTEN (1900/B/CT) variant was administered to either the FVIII/VWF DKO mice or HemA mice. The half-life of FVIII-XTEN

(1900/B/CT) is compared.

[0047] Figure 10. FVIII activity of FVIIIFc (hollow triangle), FVIII169:Fc (filled circle), and FVIII 169 :VWF31 (hollow triangle) in mouse DKO plasma measured by chromogenic assay. FVIII :Fc contains a dual-chain FVIII (Heavy chain and Light chain) fused to an Fc dimer (i.e., monomer-dimer hybrid). FVIII 169 is described above (containing AE288 in the B domain, immediately downstream of amino acid 745 corresponding to mature FVIII sequence). FVIII169:Fc contains FVIII169 fused to an Fc dimer. FVIII 169 :VWF31 contains VWF31 in addition to the Fc dimer, FVIII 169 fused to the first Fc region and VWF31 fused to the second Fc region, wherein the first Fc region and the second Fc region form a covalent bond, e.g., one or more disulfide bonds.

[0048] Figure 11. Effects of Fc, XTEN, and VWF-DO3 fragments on FVIII half- life extension. BDD-FVIII (REFACTO®) (square), FVIIFc (circle), FVIII169/Fc (triangle), and FVIII 169 /VWF031 (inverted triangle) were administered to FVIII and VWF double knockout (DKO) mice. The FVIII activity was measured by chromogenic assay, and the half-life was calculated using the WinNonlin-Phoenix program. X-axis shows time, and the Y-axis shows the FVIII plasma activity in mU/mL.

[0049] Figure 12A-C. Effects of different XTENs in rFVIII-XTEN/VWF

heterodimer in HemA mice. Figure 12A shows the FVIII plasma activity normalized to 5 min value (%) of two XTENs inserted immediately downstream of residues 1900 and 1656 corresponding to mature FVIII sequence {i.e., FVIII- 195 (dual chain FVIII isoform) and FVIII- 199 (single chain FVIII isoform)), compared to FVIII-169 containing an XTEN immediately downstream of residue 745 corresponding to mature FVIII sequence. FVIII- 169/VWF-031 (filled circle), FVIII- 199/VWF-031 (filled square), and FVIII- 195/VWF031 (hollow square) were administered in HemA mice to measure the FVIII plasma activity. Figure 12B shows the half-life extension effect of the second XTEN insertion

immediately downstream of residues 403 (A2 domain) and 745 (B domain) {i.e., FVIII-203) and residues 745 (B domain) and 1900 (A3 domain) (FVIII-204) corresponding to mature FVIII sequence compared to FVIII-169 (an XTEN insertion in B domain only). FVIII-204/VWF031 (filled triangle), FVIII- 169/VWF-031 (filled circle), FVIII-203/VWF-031 (filled square), and scBDD- FVIII (hollow diamond) were administered to HemA mice. The X-axis shows FVIII plasma activity normalized to 5 min value (%), and the y-axis shows time in hours. Figure 12C shows the half-life extension effect of the two XTEN insertions immediately downstream of residues 18 (A 1 domain) and 745 (B domain) {i.e., FVIII-205) compared to FVIII-169 (a single XTEN insertion in the B domain) and single chain FVIII without any Fc regions or any XTENs (i.e., FVIII-207). Figure 12C additionally shows the half-life extension effect of three XTEN insertions incorporated immediately downstream of residues 26 (Al domain), 1656 (A3 domain), and 1900 (A3 domain) (i.e., FVIII-201) compared to FVIII- 169 (a single XTEN insertion immediately downstream of residue 745). . FVIII-205 /VWF-031 (filled square), FVIII-20 l/VWF-031 (inverted triangle), FVIII- 169/VWF-031 (filled circle), and FVIII-207 (hollow diamond) were administered to HemA mice. The FVIII plasma activity normalized to 5 min value (%) (X-axis) was measured over time in hours (Y-axis).

[0050] Figure 13. FVIII activity of rFVIII-XTEN/VWF-XTEN heterodimer in

FVIII/VWF DKO mice. FVIII activity of plasma samples was analyzed by FVIII chromogenic assay, and the regression curve of plasma FVIII activity (X-axis) as a function of time (Y-axis) was plotted. FVIII- 155 (scFVIIIFc without any XTENs) was co-expressed with VWF-034 (VWF-Fc with AE 288 XTEN plus a 35 residue thrombin cleavable linker). The half-life of FVIII- 155 /VWF-034 was compared with that of FVIII- 169/VWF-031, which has a AE 288 XTEN inserted into the B domain junction (immediately downstream of residue 745

corresponding to mature FVIII polypeptide) of FVIII.

[0051] Figure 14A-H. Schematic diagrams of various rFVIII-XTEN/VWF

constructs. These constructs are also described in other sections herein. Fig. 14A shows single chain B domain deleted FVIII protein (sometimes indicated herein as scBDD-FVIII). The scBDD-FVIII constructs contain two substitutions at residues 1645 and 1648 from Arg to Ala. Figure 14B shows two polypeptide chain construct (FVIII 155 /VWF031), the first chain comprising single chain FVIII linked to an Fc region without any XTENS and the second chain comprising the VWF D'D3 fragment linked to an Fc region. This construct is used as a control. Fig. 14C shows two polypeptide chain construct (FVIII 199 /VWF031), the first chain comprising single chain FVIII linked to an Fc region, in which a first XTEN is inserted immediately downstream of residue 1900 corresponding to mature FVIII sequence and a second XTEN is inserted immediately downstream of residue 1656 corresponding to mature FVIII sequence, and the second chain comprising the VWF D'D3 fragment linked to an Fc region. Fig. 14D shows two polypeptide chain construct (FVIII201/VWF031), the first chain comprising single chain FVIII protein linked to an Fc region, in which a first XTEN is inserted immediately downstream of residue 26 corresponding to mature FVIII sequence, a second XTEN is inserted immediately downstream of residue 1656 corresponding to mature FVIII sequence, and a third XTEN is inserted immediately downstream of residue 1900 corresponding to mature FVIII sequence ,and the second chain comprising the VWF D'D3 fragment linked to an Fc region. Fig. 14E shows two polypeptide chain constructs (FVIII 169 /VWF031), the first chain comprising single chain FVIII protein linked to an Fc region, in which an XTEN is inserted immediately downstream of residue 745 (indicated as "B") corresponding to mature FVIII sequence, and the second chain comprising the VWF D'D3 fragment linked to an Fc region. Fig. 14F shows two polypeptide chain construct

(FVIII203/VWF031), the first chain comprising single chain FVIII protein, in which a first XTEN is inserted at residue 745 ("B") corresponding to mature FVIII sequence and a second XTEN is inserted at residue 1900 corresponding to mature FVIII sequence, and the second chain comprising the VWF D'D3 fragment linked to an Fc region. Fig. 14G shows two polypeptide chain construct

(FVIII204/VWF031), the first chain comprising single chain FVIII protein linked to an Fc region, in which a first XTEN is inserted immediately downstream of residue 403 corresponding to mature FVIII sequence and a second XTEN is inserted immediately downstream of residue 745 ("B") corresponding to mature FVIII sequence, and a second chain comprising the VWF D'D3 fragment linked to an Fc region. Fig. 14H shows two polypeptide chain construct

(FVIII205/VWF031), the first chain comprising single chain FVIII, in which a first XTEN is inserted immediately downstream of residue 18 corresponding to mature FVIII sequence and a second XTEN is inserted immediately downstream of residue 745 ("B") corresponding to mature FVIII sequence, and the second chain comprising the VWF D'D3 fragment linked to an Fc region.

Figure 15. FVIII activity of rFVIII-XTEN/VWF and BDD-F VIII in

FVIII/VWF DKO mice. FVIII activity of plasma samples was analyzed by FVIII chromogenic assay, and the regression curve of plasma FVIII activity (X-axis) as a function of time (Y-axis) was plotted. The half-life of rFVIII-XTEN/VWF (FVIII-205/VWF-031) was compared with that of BDD-FVIII and rFVIIIFc. [0053] Figure 16. Efficacy of FVIII-XTEN-Fc:VWF-Fc heterodimers in HemA mice using tail clip bleeding model. The HemA mice tail clip bleeding model was used to compare the efficacy of FVIII169/VWF034, FVIII205/VWF031, and BDD-FVIII. The median blood loss in ml for 200 IU/kg of FVIII169/VWF034 and FVIII205/VWF031 is compared with 200 IU/kg of BDD-FVIII, 65 IU/kg of BDD-FVIII, 20 IU/kg of BDD-FVIII, and vehicle.

DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS

[0054] It is to be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "a nucleotide sequence," is understood to represent one or more nucleotide sequences. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0055] The term "polynucleotide" or "nucleotide" is intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA (pDNA). In certain embodiments, a polynucleotide comprises a

conventional phosphodiester bond or a non-conventional bond {e.g., an amide bond, such as found in peptide nucleic acids (PNA)). The term "nucleic acid" refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide. By "isolated" nucleic acid or polynucleotide is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, a recombinant polynucleotide encoding a Factor VIII polypeptide contained in a vector is considered isolated for the purposes of the present invention. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) from other polynucleotides in a solution.

Isolated RNA molecules include in vivo or in vitro RNA transcripts of

polynucleotides of the present invention. Isolated polynucleotides or nucleic acids according to the present invention further include such molecules produced synthetically. In addition, a polynucleotide or a nucleic acid can include regulatory elements such as promoters, enhancers, ribosome binding sites, or transcription termination signals. [0056] As used herein, a "coding region" or "coding sequence" is a portion of polynucleotide which consists of codons translatable into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is typically not translated into an amino acid, it may be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region. The boundaries of a coding region are typically determined by a start codon at the 5' terminus, encoding the amino terminus of the resultant polypeptide, and a translation stop codon at the 3' terminus, encoding the carboxyl terminus of the resulting polypeptide. Two or more coding regions of the present invention can be present in a single

polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. It follows, then, that a single vector can contain just a single coding region, or comprise two or more coding regions, e.g., a single vector can separately encode a binding domain-A and a binding domain-B as described below. In addition, a vector, polynucleotide, or nucleic acid of the invention can encode heterologous coding regions, either fused or unfused to a nucleic acid encoding a binding domain of the invention.

Heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.

[0057] Certain proteins secreted by mammalian cells are associated with a

secretory signal peptide which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those of ordinary skill in the art are aware that signal peptides are generally fused to the N-terminus of the polypeptide, and are cleaved from the complete or "full-length" polypeptide to produce a secreted or "mature" form of the polypeptide. In certain embodiments, a native signal peptide or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it. Alternatively, a heterologous mammalian signal peptide, e.g. , a human tissue plasminogen activator (TP A) or mouse β-glucuronidase signal peptide, or a functional derivative thereof, can be used.

[0058] The term "downstream" refers to a nucleotide sequence that is located 3' to a reference nucleotide sequence. In certain embodiments, downstream nucleotide sequences relate to sequences that follow the starting point of transcription. For example, the translation initiation codon of a gene is located downstream of the start site of transcription.

[0059] The term "upstream" refers to a nucleotide sequence that is located 5' to a reference nucleotide sequence. In certain embodiments, upstream nucleotide sequences relate to sequences that are located on the 5' side of a coding region or starting point of transcription. For example, most promoters are located upstream of the start site of transcription.

[0060] As used herein, the term "regulatory region" refers to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding region, and which influence the transcription, RNA processing, stability, or translation of the associated coding region. Regulatory regions may include promoters, translation leader sequences, introns,

polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem-loop structures. If a coding region is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.

[0061] A polynucleotide which encodes a gene product, e.g., a polypeptide, can include a promoter and/or other transcription or translation control elements operably associated with one or more coding regions. In an operable association a coding region for a gene product, e.g., a polypeptide, is associated with one or more regulatory regions in such a way as to place expression of the gene product under the influence or control of the regulatory region(s). For example, a coding region and a promoter are "operably associated" if induction of promoter function results in the transcription of mRNA encoding the gene product encoded by the coding region, and if the nature of the linkage between the promoter and the coding region does not interfere with the ability of the promoter to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can also be operably associated with a coding region to direct gene product expression. [0062] A variety of transcription control regions are known to those skilled in the art. These include, without limitation, transcription control regions which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with intron-A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit β-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as lymphokine -inducible promoters (e.g., promoters inducible by interferons or interleukins).

[0063] Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence).

[0064] The term "expression" as used herein refers to a process by which a

polynucleotide produces a gene product, for example, an RNA or a polypeptide. It includes without limitation transcription of the polynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) or any other RNA product, and the translation of an mRNA into a polypeptide. Expression produces a "gene product." As used herein, a gene product can be either a nucleic acid, e.g. , a messenger RNA produced by transcription of a gene, or a polypeptide which is translated from a transcript. Gene products described herein further include nucleic acids with post transcriptional modifications, e.g. , polyadenylation or splicing, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, or proteolytic cleavage.

[0065] A "vector" refers to any vehicle for the cloning of and/or transfer of a

nucleic acid into a host cell. A vector may be a replicon to which another nucleic acid segment may be attached so as to bring about the replication of the attached segment. A "replicon" refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of replication in vivo, i.e., capable of replication under its own control. The term "vector" includes both viral and nonviral vehicles for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo. A large number of vectors are known and used in the art including, for example, plasmids, modified eukaryotic viruses, or modified bacterial viruses. Insertion of a polynucleotide into a suitable vector can be accomplished by ligating the appropriate polynucleotide fragments into a chosen vector that has complementary cohesive termini.

[0066] Vectors may be engineered to encode selectable markers or reporters that provide for the selection or identification of cells that have incorporated the vector. Expression of selectable markers or reporters allows identification and/or selection of host cells that incorporate and express other coding regions contained on the vector. Examples of selectable marker genes known and used in the art include: genes providing resistance to ampicillin, streptomycin, gentamycin, kanamycin, hygromycin, bialaphos herbicide, sulfonamide, and the like; and genes that are used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase gene, and the like. Examples of reporters known and used in the art include: lucif erase (Luc), green fluorescent protein (GFP),

chloramphenicol acetyltransferase (CAT), -galactosidase (LacZ), -glucuronidase (Gus), and the like. Selectable markers may also be considered to be reporters.

[0067] The term "plasmid" refers to an extra-chromosomal element often

carrying a gene that is not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear, circular, or supercoiled, of a single- or double- stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell.

[0068] Eukaryotic viral vectors that can be used include, but are not limited to, adenovirus vectors, retrovirus vectors, adeno-associated virus vectors, and poxvirus, e.g., vaccinia virus vectors, baculovirus vectors, or herpesvirus vectors. Non-viral vectors include plasmids, liposomes, electrically charged lipids

(cytofectins), DNA-protein complexes, and biopolymers. [0069] A "cloning vector" refers to a "replicon," which is a unit length of a nucleic acid that replicates sequentially and which comprises an origin of replication, such as a plasmid, phage or cosmid, to which another nucleic acid segment may be attached so as to bring about the replication of the attached segment. Certain cloning vectors are capable of replication in one cell type, e.g., bacteria and expression in another, e.g., eukaryotic cells. Cloning vectors typically comprise one or more sequences that can be used for selection of cells comprising the vector and/or one or more multiple cloning sites for insertion of nucleic acid sequences of interest.

[0070] The term "expression vector" refers to a vehicle designed to enable the expression of an inserted nucleic acid sequence following insertion into a host cell. The inserted nucleic acid sequence is placed in operable association with regulatory regions as described above.

[0071] Vectors are introduced into host cells by methods well known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a DNA vector transporter.

[0072] "Culture," "to culture" and "culturing," as used herein, means to incubate cells under in vitro conditions that allow for cell growth or division or to maintain cells in a living state. "Cultured cells," as used herein, means cells that are propagated in vitro.

[0073] As used herein, the term "polypeptide" is intended to encompass a singular

"polypeptide" as well as plural "polypeptides," and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of "polypeptide," and the term "polypeptide" can be used instead of, or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation,

phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide can be derived from a natural biological source or produced recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It can be generated in any manner, including by chemical synthesis.

[0074] An "isolated" polypeptide or a fragment, variant, or derivative thereof refers to a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an isolated polypeptide can simply be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purpose of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0075] Also included in the present invention are fragments or variants of

polypeptides, and any combination thereof. The term "fragment" or "variant" when referring to polypeptide binding domains or binding molecules of the present invention include any polypeptides which retain at least some of the properties (e.g., FcRn binding affinity for an FcRn binding domain or Fc variant, coagulation activity for an FVIII variant, or FVIII binding activity for the VWF fragment) of the reference polypeptide. Fragments of polypeptides include proteolytic fragments, as well as deletion fragments, in addition to specific antibody fragments discussed elsewhere herein, but do not include the naturally occurring full-length polypeptide (or mature polypeptide). Variants of polypeptide binding domains or binding molecules of the present invention include fragments as described above, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants can be naturally or non-naturally occurring. Non-naturally occurring variants can be produced using art-known mutagenesis techniques. Variant polypeptides can comprise conservative or non-conservative amino acid substitutions, deletions or additions.

[0076] The term "VWF fragment" or "VWF fragments" used herein means any

VWF fragments that interact with FVIII and retain at least one or more properties that are normally provided to FVIII by full-length VWF, e.g., preventing premature activation to FVIIIa, preventing premature proteolysis, preventing association with phospholipid membranes that could lead to premature clearance, preventing binding to FVIII clearance receptors that can bind naked FVIII but not VWF-bound FVIII, and/or stabilizing the FVIII heavy chain and light chain interactions.

[0077] A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.

Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g. , alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g. , tyrosine, phenylalanine, tryptophan, histidine). Thus, if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the substitution is considered to be conservative. In another embodiment, a string of amino acids can be

conservatively replaced with a structurally similar string that differs in order and/or composition of side chain family members.

[0078] As known in the art, "sequence identity" between two polypeptides is determined by comparing the amino acid sequence of one polypeptide to the sequence of a second polypeptide. When discussed herein, whether any particular polypeptide is at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to another polypeptide can be determined using methods and computer programs/software known in the art such as, but not limited to, the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 5371 1). BESTFIT uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology between two sequences. When using BESTFIT or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full-length of the reference polypeptide sequence and that gaps in homology of up to 5% of the total number of amino acids in the reference sequence are allowed.

[0079] As used herein, an "amino acid corresponding to" or an "equivalent amino acid" in a VWF sequence or a FVIII protein sequence is identified by alignment to maximize the identity or similarity between a first VWF or FVIII sequence and a second VWF or FVIII sequence. The number used to identify an equivalent amino acid in a second VWF or FVIII sequence is based on the number used to identify the corresponding amino acid in the first VWF or FVIII sequence.

[0080] As used herein, the term "insertion site" refers to a position in a FVIII polypeptide, or fragment, variant, or derivative thereof, which is immediately upstream of the position at which a heterologous moiety can be inserted. An "insertion site" is specified as a number, the number being the number of the amino acid in mature native FVIII (SEQ ID NO: 4) to which the insertion site corresponds, which is immediately N-terminal to the position of the insertion. For example, the phrase "a3 comprises an XTEN at an insertion site which

corresponds to amino acid 1656 of SEQ ID NO: 4" indicates that the heterologous moiety is located between two amino acids corresponding to amino acid 1656 and amino acid 1657 of SEQ ID NO: 4.

[0081] The phrase "immediately downstream of an amino acid" as used herein refers to position right next to the terminal carboxyl group of the amino acid. Similarly, the phrase "immediately upstream of an amino acid" refers to the position right next to the terminal amine group of the amino acid. Therefore, the phrase "between two amino acids of an insertion site" as used herein refers to a position in which an XTEN or any other polypeptide is inserted between two adjacent amino acids. Thus, the phrases "inserted immediately downstream of an amino acid" and "inserted between two amino acids of an insertion site" are used synonymously with "inserted at an insertion site."

[0082] The terms "inserted," "is inserted," "inserted into" or grammatically related terms, as used herein refers to the position of an XTEN in a chimeric polypeptide relative to the analogous position in native mature human FVIII. As used herein the terms refer to the characteristics of the recombinant FVIII polypeptide relative to native mature human FVIII, and do not indicate, imply or infer any methods or process by which the chimeric polypeptide was made. For example, in reference to a chimeric polypeptide provided herein, the phrase "an XTEN is inserted into immediately downstream of residue 745 of the FVIII polypeptide" means that the chimeric polypeptide comprises an XTEN immediately downstream of an amino acid which corresponds to amino acid 745 in native mature human FVIII, e.g., bounded by amino acids corresponding to amino acids 745 and 746 of native mature human FVIII.

[0083] A "fusion" or "chimeric" protein comprises a first amino acid sequence linked to a second amino acid sequence with which it is not naturally linked in nature. The amino acid sequences which normally exist in separate proteins can be brought together in the fusion polypeptide, or the amino acid sequences which normally exist in the same protein can be placed in a new arrangement in the fusion polypeptide, e.g. , fusion of a Factor VIII domain of the invention with an Ig Fc domain. A fusion protein is created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship. A chimeric protein can further comprises a second amino acid sequence associated with the first amino acid sequence by a covalent, non-peptide bond or a non-covalent bond.

[0084] As used herein, the term "half-life" refers to a biological half-life of a particular polypeptide in vivo. Half-life may be represented by the time required for half the quantity administered to a subject to be cleared from the circulation and/or other tissues in the animal. When a clearance curve of a given polypeptide is constructed as a function of time, the curve is usually biphasic with a rapid a- phase and longer β-phase. The a-phase typically represents an equilibration of the administered Fc polypeptide between the intra- and extra-vascular space and is, in part, determined by the size of the polypeptide. The β-phase typically represents the catabolism of the polypeptide in the intravascular space. In some

embodiments, FVIII and chimeric proteins comprising FVIII are monophasic, and thus do not have an alpha phase, but just the single beta phase. Therefore, in certain embodiments, the term half-life as used herein refers to the half-life of the polypeptide in the β-phase. The typical β-phase half-life of a human antibody in humans is 21 days. [0085] The term "linked" as used herein refers to a first amino acid sequence or nucleotide sequence covalently or non-covalently joined to a second amino acid sequence or nucleotide sequence, respectively. The first amino acid or nucleotide sequence can be directly joined or juxtaposed to the second amino acid or nucleotide sequence or alternatively an intervening sequence can covalently join the first sequence to the second sequence. The term "linked" means not only a fusion of a first amino acid sequence to a second amino acid sequence at the C- terminus or the N-terminus, but also includes insertion of the whole first amino acid sequence (or the second amino acid sequence) into any two amino acids in the second amino acid sequence (or the first amino acid sequence, respectively). In one embodiment, the first amino acid sequence can be linked to a second amino acid sequence by a peptide bond or a linker. The first nucleotide sequence can be linked to a second nucleotide sequence by a phosphodiester bond or a linker. The linker can be a peptide or a polypeptide (for polypeptide chains) or a nucleotide or a nucleotide chain (for nucleotide chains) or any chemical moiety (for both polypeptide and polynucleotide chains). The term "linked" is also indicated by a hyphen (-).

[0086] As used herein the term "associated with" refers to a covalent or non- covalent bond formed between a first amino acid chain and a second amino acid chain. In one embodiment, the term "associated with" means a covalent, non- peptide bond or a non-covalent bond. This association can be indicated by a colon, i.e., (:). In another embodiment, it means a covalent bond except a peptide bond. For example, the amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a thiol group on a second cysteine residue. In most naturally occurring IgG molecules, the CHI and CL regions are associated by a disulfide bond and the two heavy chains are associated by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system

(position 226 or 229, EU numbering system). Examples of covalent bonds include, but are not limited to, a peptide bond, a metal bond, a hydrogen bond, a disulfide bond, a sigma bond, a pi bond, a delta bond, a glycosidic bond, an agnostic bond, a bent bond, a dipolar bond, a Pi backbond, a double bond, a triple bond, a quadruple bond, a quintuple bond, a sextuple bond, conjugation,

hyperconjugation, aromaticity, hapticity, or antibonding. Non-limiting examples of non-covalent bond include an ionic bond (e.g., cation-pi bond or salt bond), a metal bond, an hydrogen bond (e.g., dihydrogen bond, dihydrogen complex, low- barrier hydrogen bond, or symmetric hydrogen bond), van der Walls force, London dispersion force, a mechanical bond, a halogen bond, aurophilicity, intercalation, stacking, entropic force, or chemical polarity.

[0087] The term "monomer-dimer hybrid" used herein refers to a chimeric protein comprising a first polypeptide chain and a second polypeptide chain, which are associated with each other by a disulfide bond, wherein the first chain comprises a clotting factor, e.g., Factor VIII, and a first Fc region and the second chain comprises, consists essentially of, or consists of a second Fc region without the clotting factor. The monomer-dimer hybrid construct thus is a hybrid comprising a monomer aspect having only one clotting factor and a dimer aspect having two Fc regions.

[0088] As used herein, the term "cleavage site" or "enzymatic cleavage site" refers to a site recognized by an enzyme. Certain enzymatic cleavage sites comprise an intracellular processing site. In one embodiment, a polypeptide has an enzymatic cleavage site cleaved by an enzyme that is activated during the clotting cascade, such that cleavage of such sites occurs at the site of clot formation. Exemplary such sites include, e.g., those recognized by thrombin, Factor XIa or Factor Xa. Exemplary FXIa cleavage sites include, e.g., TQSFNDFTR (SEQ ID NO: 45) and SVSQTSKLTR (SEQ ID NO: 46). Exemplary thrombin cleavage sites include, e.g., DFLAEGGGVR (SEQ ID NO: 47), TTKIKPR (SEQ ID NO: 48), LVPRG (SEQ ID NO: 49) and ALRPR (amino acids 1 to 5 of SEQ ID NO: 50). Other enzymatic cleavage sites are known in the art.

[0089] As used herein, the term "processing site" or "intracellular processing site" refers to a type of enzymatic cleavage site in a polypeptide which is a target for enzymes that function after translation of the polypeptide. In one embodiment, such enzymes function during transport from the Golgi lumen to the trans-Golgi compartment. Intracellular processing enzymes cleave polypeptides prior to secretion of the protein from the cell. Examples of such processing sites include, e.g., those targeted by the PACE/furin (where PACE is an acronym for Paired basic Amino acid Cleaving Enzyme) family of endopeptidases. These enzymes are localized to the Golgi membrane and cleave proteins on the carboxyterminal side of the sequence motif Arg-[any residue]-(Lys or Arg)-Arg. As used herein the "furin" family of enzymes includes, e.g., PCSK1 (also known as PCl/Pc3), PCSK2 (also known as PC2), PCSK3 (also known as furin or PACE), PCSK4 (also known as PC4), PCSK5 (also known as PC5 or PC6), PCSK6 (also known as PACE4), or PCSK7 (also known as PC7/LPC, PC8, or SPC7). Other processing sites are known in the art.

[0090] In constructs that include more than one processing or cleavage site, it will be understood that such sites may be the same or different.

[0091] The term "Furin" refers to the enzymes corresponding to EC No. 3.4.21.75.

Furin is subtilisin-like proprotein convertase, which is also known as PACE (Paired basic Amino acid Cleaving Enzyme). Furin deletes sections of inactive precursor proteins to convert them into biologically active proteins. During its intracellular transport, pro-peptide of VWF can be cleaved from mature VWF molecule by a Furin enzyme. In some embodiments, Furin cleaves the D1D2 from the D'D3 of VWF. In other embodiments, a nucleotide sequence encoding Furin can be expressed together with the nucleotide sequence encoding a VWF fragment so that D1D2 domains can be cleaved off intracellularly by Furin.

[0092] In constructs that include more than one processing or cleavage site, it will be understood that such sites may be the same or different.

[0093] A "processable linker" as used herein refers to a linker comprising at least one intracellular processing site, which is described elsewhere herein.

[0094] Hemostatic disorder, as used herein, means a genetically inherited or

acquired condition characterized by a tendency to hemorrhage, either

spontaneously or as a result of trauma, due to an impaired ability or inability to form a fibrin clot. Examples of such disorders include the hemophilias. The three main forms are hemophilia A (factor VIII deficiency), hemophilia B (factor IX deficiency or "Christmas disease") and hemophilia C (factor XI deficiency, mild bleeding tendency). Other hemostatic disorders include, e.g., Von Willebrand disease, Factor XI deficiency (PTA deficiency), Factor XII deficiency,

deficiencies or structural abnormalities in fibrinogen, prothrombin, Factor V, Factor VII, Factor X or factor XIII, Bernard-Soulier syndrome, which is a defect or deficiency in GPIb. GPIb, the receptor for VWF, can be defective and lead to lack of primary clot formation (primary hemostasis) and increased bleeding tendency), and thrombasthenia of Glanzman and Naegeli (Glanzmann

thrombasthenia). In liver failure (acute and chronic forms), there is insufficient production of coagulation factors by the liver; this may increase bleeding risk.

[0095] The chimeric molecules of the invention can be used prophylactically. As used herein the term "prophylactic treatment" refers to the administration of a molecule prior to a bleeding episode. In one embodiment, the subject in need of a general hemostatic agent is undergoing, or is about to undergo, surgery. The chimeric protein of the invention can be administered prior to or after surgery as a prophylactic. The chimeric protein of the invention can be administered during or after surgery to control an acute bleeding episode. The surgery can include, but is not limited to, liver transplantation, liver resection, dental procedures, or stem cell transplantation.

[0096] The chimeric protein of the invention is also used for on-demand

treatment. The term "on-demand treatment" refers to the administration of a chimeric molecule in response to symptoms of a bleeding episode or before an activity that may cause bleeding. In one aspect, the on-demand treatment can be given to a subject when bleeding starts, such as after an injury, or when bleeding is expected, such as before surgery. In another aspect, the on-demand treatment can be given prior to activities that increase the risk of bleeding, such as contact sports.

[0097] As used herein the term "acute bleeding" refers to a bleeding episode

regardless of the underlying cause. For example, a subject may have trauma, uremia, a hereditary bleeding disorder (e.g., factor VII deficiency) a platelet disorder, or resistance owing to the development of antibodies to clotting factors.

[0098] Treat, treatment, treating, as used herein refers to, e.g., the reduction in severity of a disease or condition; the reduction in the duration of a disease course; the amelioration of one or more symptoms associated with a disease or condition; the provision of beneficial effects to a subject with a disease or condition, without necessarily curing the disease or condition, or the prophylaxis of one or more symptoms associated with a disease or condition. In one embodiment, the term "treating" or "treatment" means maintaining a FVIII trough level at least about 1 IU/dL, 2 IU/dL, 3 IU/dL, 4 IU/dL, 5 IU/dL, 6 IU/dL, 7 IU/dL, 8 IU/dL, 9 IU/dL, 10 IU/dL, 11 IU/dL, 12 IU/dL, 13 IU/dL, 14 IU/dL, 15 IU/dL, 16 IU/dL, 17 IU/dL, 18 IU/dL, 19 IU/dL, or 20 IU/dL in a subject by administering a chimeric protein or a VWF fragment of the invention. In another embodiment, treating or treatment means maintaining a FVIII trough level between about 1 and about 20 IU/dL, about 2 and about 20 IU/dL, about 3 and about 20 IU/dL, about 4 and about 20 IU/dL, about 5 and about 20 IU/dL, about 6 and about 20 IU/dL, about 7 and about 20 IU/dL, about 8 and about 20 IU/dL, about 9 and about 20 IU/dL, or about 10 and about 20 IU/dL. Treatment or treating of a disease or condition can also include maintaining FVIII activity in a subject at a level comparable to at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the FVIII activity in a non-hemophiliac subject. The minimum trough level required for treatment can be measured by one or more known methods and can be adjusted (increased or decreased) for each person.

CHIMERIC PROTEINS

The present invention is directed to extending the half-life of a Factor VIII protein using a VWF fragment and an XTEN sequence by preventing or inhibiting a FVIII half-life limiting factor, i.e., endogenous VWF, from associating with the FVIII protein. Endogenous VWF associates with about 95% to about 98%> of FVIII in non-covalent complexes. While endogenous VWF is a FVIII half-life limiting factor, endogenous VWF bound to a FVIII protein is also known to protect FVIII in various ways. For example, full length VWF (as a multimer having about 250 kDa) can protect FVIII from protease cleavage and FVIII activation, stabilize the FVIII heavy chain and/or light chain, and prevent clearance of FVIII by scavenger receptors. But, at the same time, endogenous VWF limits the FVIII half-life by preventing pinocytosis and by clearing FVIII - VWF complex from the system through the VWF clearance pathway. It is believed, while not bound by a theory, that endogenous VWF is a half-life limiting factor that prevents the half-life of a FVIII protein fused to a half-life extender from being longer than about two-fold that of wild-type FVIII. Therefore, the present invention is directed to preventing or inhibiting interaction between endogenous VWF and a FVIII protein using a VWF fragment, thereby increasing a half-life of the FVIII protein by using an XTEN sequence alone or an XTEN sequence in combination with an Ig constant region or a portion thereof. The XTEN sequence can be linked to the FVIII protein or the VWF fragment. The FVIII protein associated with the VWF fragment is thus cleared from the circulation more slowly by one or more VWF clearance receptors and then can have the full half-life extension of the XTEN sequence or the XTEN sequence in combination of the Ig constant region, as compared to wild type FVIII or a FVIII protein without the VWF fragment.

[0100] In one embodiment, a VWF fragment is associated (or linked) with the

FVIII protein by a covalent or a non-covalent bond. In some instances, however, the physical blockage or chemical association (e.g., non-covalent bonding) between the VWF fragment and the FVIII protein may not be strong enough to provide a stable complex comprising the FVIII protein and the VWF fragment in the presence of endogenous VWF. For example, a VWF fragment forming a non- covalent bond with a FVIII protein without any other connections may readily be dissociated in vivo from the FVIII protein in the presence of endogenous VWF, replacing the VWF fragment (e.g., recombinant VWF, i.e., rVWF) with endogenous VWF. Therefore, the FVIII protein non-covalently bound to endogenous VWF would undergo the VWF clearance pathway and be readily cleared from the system. In order to prevent the dissociation of the VWF fragment with the FVIII protein, in some embodiments, the association or linkage between the FVIII protein and the VWF fragment is a covalent bond, e.g., a peptide bond, one or more amino acids, or a disulfide bond. In certain

embodiments, the association (i.e., linkage) between the adjunct moiety and the FVIII protein is a peptide bond or a linker between the FVIII protein and the VWF fragment ("FVIII/VWF linker"). Non- limiting examples of the linker are described elsewhere herein. In some embodiments, the VWF fragment is a polypeptide comprising, consisting essentially of, or consisting of at least about 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, or 4000 amino acids. Non- limiting examples of the VWF fragment are described elsewhere herein.

[0101] In certain embodiments, the VWF fragment chemically (e.g., non- covalently) binds to or physically blocks one or more VWF binding sites on a FVIII protein. The VWF binding site on a FVIII protein is located within the A3 domain or the C2 domain of the FVIII protein. In still other embodiments, the VWF binding site on a FVIII protein is located within the A3 domain and C2 domain. For example, the VWF binding site on a FVIII protein can correspond to amino acids 1669 to 1689 and/or 2303 to 2332 of SEQ ID NO: 4 [full-length mature FVIII].

[0102] The invention also provides a chimeric protein (comprising a FVIII protein and a VWF fragment) further comprising one or more XTEN sequences, which provide additional half-life extension properties. The one or more XTEN sequences can be inserted within the FVIII protein or the VWF fragment or linked to the N-terminus or the C-terminus of the FVIII protein or the VWF fragment. The invention also includes a FVIII protein linked to an XTEN sequence (a first half-life extending moiety) and an Ig constant region or a portion thereof (a second half-life extending moiety) so that the two half-life extending moieties extend the half-life of the FVIII protein through two different mechanisms.

[0103] In some embodiments, a chimeric protein comprises a FVIII protein linked to a first Ig constant region or a portion thereof (e.g., a first FcRn binding partner), a VWF fragment linked to a second Ig constant region or a portion thereof (e.g., a second FcRn binding partner), and one or more XTEN sequences inserted or linked to the FVIII protein or the VWF fragment, wherein the VWF fragment prevents the FVIII half-life limiting factor (e.g., endogenous VWF) from binding to the FVIII protein, wherein the first and second Ig constant regions or portions thereof forms a covalent bond, e.g., a disulfide bond, and the one or more XTEN sequences extends the half-life of the FVIII protein.

[0104] In certain embodiments, a chimeric protein of the invention comprises a

FVIII protein linked to a VWF fragment by an optional linker (i.e., FVIII/VWF linker) and one or more XTEN sequences inserted or linked to the FVIII protein or the VWF fragment, wherein the VWF fragment prevents the FVIII half-life limiting factor (e.g., endogenous VWF) from binding to the FVIII protein and the one or more XTEN sequences extends the half-life of the FVIII protein. In one aspect, the optional linker (FVIII/VWF linker) comprises a sortase recognition motif. In another aspect, the optional linker (FVIII/VWF linker) comprises a cleavable site. Examples of the cleavage linker (i.e., linker containing one or more cleavage site) are described elsewhere herein. [0105] The chimeric protein of the present invention includes, but is not limited to:

(1) a VWF fragment comprising a D' domain and a D3 domain, an XTEN sequence, and FVIII, wherein the XTEN sequence is linked to the VWF fragment;

(2) a FVIII protein, an XTEN sequence, and an Ig constant region or a portion thereof, wherein the FVIII protein is linked to an XTEN sequence and the Ig constant region or a portion thereof, or

(3) a FVIII protein, an XTEN sequence, and a VWF fragment, wherein the XTEN sequence is linked to the FVIII protein at the C-terminus or N-terminus or inserted immediately downstream of one or more amino acids (e.g., one or more XTEN insertion sites) of FVIII, and the VWF fragment and the FVIII protein are associated with each other.

(1) Von Willebrand Factor (VWF) fragment linked to XTEN, and FVIII

[0106] The present invention is directed to a chimeric protein comprising (i) a

VWF fragment comprising a D' domain and a D3 domain of VWF, (ii) an XTEN sequence, and (iii) a FVIII protein, wherein (i), (ii), and (iii) are linked to or associated with each other. The VWF fragment linked to the XTEN sequence, as a part of a chimeric protein in the present invention, associates with the FVIII protein, thus preventing or inhibiting interaction between endogenous VWF and the FVIII protein. In certain embodiments, the VWF fragment, which is capable of preventing or inhibiting binding of the FVIII protein with endogenous VWF, can at the same time have at least one VWF-like FVIII protecting property.

Examples of the VWF-like FVIII protecting properties include, but are not limited to, protecting FVIII from protease cleavage and FVIII activation, stabilizing the FVIII heavy chain and/or light chain, and preventing clearance of FVIII by scavenger receptors. As a result, the VWF fragment can prevent clearance of the FVIII protein through the VWF clearance pathway, thus reducing clearance of FVIII from the circulatory system. In some embodiments, the VWF fragments of the present invention bind to or are associated with a FVIII protein and/or physically or chemically block the VWF binding site on the FVIII protein. The FVIII protein associated with the VWF fragment is thus cleared from the circulation more slowly, as compared to wild type FVIII or FVIII not associated with the VWF fragment. [0107] In one embodiment, the invention is directed to a chimeric protein comprising (i) a VWF fragment comprising the D' domain and the D3 domain of VWF, (ii) an XTEN sequence, and (iii) a FVIII protein, wherein the XTEN sequence is linked to the VWF fragment (e.g., (al) V-X or (a2) X-V, wherein V comprises a VWF fragment and X comprises an XTEN sequence), and the VWF fragment is linked to or associated with the FVIII protein. In another

embodiment, the VWF fragment and the XTEN sequence can be linked by a linker (e.g., (a3) V-L-X or (a4) X-L-V) or a peptide bond. The linker can be a cleavable linker, e.g., a thrombin cleavable linker, which can be cleaved at the site of coagulation. In other embodiments, the VWF fragment, the XTEN sequence, and the FVIII protein are placed in a single polypeptide chain. In still other embodiments, the chimeric protein comprises two polypeptide chains, a first chain comprising the VWF fragment and the XTEN sequence and a second chain comprising the FVIII protein. In yet other embodiments, the chimeric protein comprises three polypeptide chains, a first chain comprising the VWF fragment and the XTEN sequence, a second chain comprising a light chain of FVIII and a third chain comprising a heavy chain of FVIII, wherein the first chain and the second chain are associated with each other (e.g., covalent bond, e.g., disulfide bond), and the second chain and the third chain are associated with each other (e.g., metal bond). In still other embodiments, the XTEN sequence can be linked to the N-terminus or the C-terminus of the VWF fragment or inserted immediately downstream of one or more amino acids in the VWF fragment.

[0108] In certain embodiments, a chimeric protein of the invention comprises a formula comprising:

(a) V-X-FVIII,

(b) FVIII-X-V,

(c) V-X:FVIII,

(d) X-V:FVIII,

(e) FVIII:V-X,

(f) FVIII:X-V, or

(a5) X-V-FVIII,

wherein V comprises a VWF fragment,

X comprises one or more XTEN sequences, FVIII comprises a FVIII protein;

(-) represents a peptide bond or one or more amino acids; and

(:) is a chemical association or a physical association. In one embodiment, (:) represents a chemical association, e.g., at least one non-peptide bond. In another embodiment, the chemical association, i.e., (:) is a covalent bond. In other embodiments, the chemical association, i.e., (:) is a non-covalent interaction, e.g., an ionic interaction, a hydrophobic interaction, a hydrophilic interaction, a Van der Waals interaction, or a hydrogen bond. In other embodiments, (:) is a non- peptide covalent bond. In still other embodiments, (:) is a peptide bond. In yet other embodiments, (:) represents a physical association between two sequences, wherein a portion of a first sequence is in close proximity to a second sequence such that the first sequence shields or blocks a portion of the second sequence from interacting with another moiety, and further that this physical association is maintained without allowing the second sequence to interact with other moieties. The orientation of the polypeptide formulas herein is listed from N-terminus (left) to C-terminus (right). For example, formula V-X-FVIII means formula NH2-V- X-FVIII -COOH. In one embodiment, the formulas described herein can comprise any additional sequences between the two moieties. For example, formula V-X- FVIII can further comprise any sequences at the N-terminus of V between V and X, between X and FVIII, or at the C-terminus of FVIII unless otherwise specified. In another embodiment, the hyphen (-) indicates a peptide bond.

In other embodiments, a chimeric protein of the invention comprises a formula comprising:

(a) V(X1)-X2-FVIII,

(b) FVIII-X2-V(X1),

(c) V(X1):FVIII,

(d) FVIII: V(X1), or

(a5) X2-V(X1)-FVIII,

wherein V(X1) comprises a VWF fragment and a first XTEN sequence (XI), wherein the XTEN sequence is inserted immediately downstream of one or more amino acids in the VWF fragment,

X2 comprises one or more optional XTEN sequences,

FVIII comprises a FVIII protein; (-) is a peptide bond or one or more amino acids; and

(:) is a chemical association or a physical association.

In some embodiments, a chimeric protein comprises (i) a VWF fragment comprising a D' domain and a D3 domain of VWF, (ii) an XTEN sequence, (iii) a FVIII protein, (iv) a first optional linker, and (v) a second optional linker, wherein the XTEN sequence is linked to the VWF fragment and/or to the FVIII protein by the linker. In certain embodiments, a chimeric protein comprises a formula comprising:

(bl) V-L1-X-L2-FVIII,

0>2) FVIII-L2-X-L1-V,

0>3) V-L1-X:FVIII,

0⁾4) X-L1-V:FVIII,

0⁾5) FVIII:V-L1-X,

0⁾6) FVIII:X-L1-V,

0⁾7) X-L1-V-L2-FVIII, or

0⁾8) FVIII-L2-V-L1-X,

wherein V comprises a VWF fragment,

X comprises one or more XTEN sequences,

FVIII comprises a FVIII protein,

LI comprises a first optional linker, e.g., a first cleavable linker,

L2 comprises a second optional linker, e.g., a second cleavable linker or an optional processable linker;

(-) is a peptide bond or one or amino acids; and

(:) is a chemical association or a physical association. In one embodiment, (:) represents a chemical association, e.g., at least one non-peptide bond. In another embodiment, the chemical association, i.e., (:) is a covalent bond. In other embodiments, the chemical association, i.e., (:) is a non-covalent interaction, e.g., an ionic interaction, a hydrophobic interaction, a hydrophilic interaction, a Van der Waals interaction, or a hydrogen bond. In other embodiments, (:) is a non- peptide covalent bond. In still other embodiments, (:) is a peptide bond. In yet other embodiments, (:) represents a physical association between two sequences, wherein a portion of a first sequence is in close proximity to a second sequence such that the first sequence shields or blocks a portion of the second sequence from interacting with another moiety, and further that this physical association is maintained without allowing the second sequence to interact with other moieties. The orientation of the polypeptide formulas herein is listed from N-terminus (left) to C-terminus (right). For example, formula (bl) V-L1-X-L2-FVIII means formula NH2-V-L1-X-L2-FVIII-COOH. In one embodiment, the formulas described herein can comprise any additional sequences between the two moieties. In another embodiment, the hyphen (-) indicates a peptide bond.

Another aspect of the present invention is to provide a FVIII chimeric protein having reduced or no interactions with a FVIII half-life limiting factor, e.g., endogenous VWF, and at the same time maximizing the half-life of the FVIII protein using an XTEN sequence (a first half-life extender) in combination with a second half-life extender or a moiety providing a covalent bond between the FVIII protein and the VWF fragment, e.g., an Ig constant region or a portion thereof. In one embodiment, a chimeric protein of the invention comprises (i) a VWF fragment comprising a D' domain and a D3 domain of VWF, (ii) an XTEN sequence, (iii) a FVIII protein, and (iv) an Ig constant region or a portion thereof (also referred to herein as F), wherein (1) the VWF fragment is linked to the XTEN sequence by an optional linker, e.g., a cleavable linker, (2) the VWF fragment is associated with or linked to the FVIII protein by an additional optional linker, e.g., a cleavable linker, and (3) the Ig constant region or a portion thereof is linked to the VWF fragment, the XTEN sequence, or the FVIII protein. In another embodiment, a chimeric protein of the invention comprises (i) a VWF fragment comprising a D' domain and a D3 domain of VWF, (ii) an XTEN sequence, (iii) a FVIII protein, (iv) an Ig constant region or a portion thereof (Fl or a first Ig constant region or a portion thereof), and (v) an additional Ig constant region or a portion thereof (F2 or a second Ig constant region or a portion thereof), wherein (1) the VWF fragment is linked to the XTEN sequence by an optional linker, e.g., a cleavable linker, (2) the XTEN sequence or the VWF fragment is linked to the Ig constant region or a portion thereof, (3) the FVIII is linked to the additional Ig constant region or a portion thereof, and (4) the Ig constant region or a portion thereof is associated with or linked to the additional Ig constant region or a portion thereof. In one embodiment, the association or linkage between the two Ig constant regions or a portion thereof is a covalent bond, e.g., a disulfide bond. In another embodiment, the association or linkage between the two Ig constant regions or a portion thereof is a processable linker, wherein the processable linker is intracellularly processed by a protease. For example, the chimeric protein comprises a formula comprising:

(g) V-L2-X-L1-F1 : FVIII-L3-F2;

GO V-L2-X-L1-F1 :F2-L3-FVni;

(i) F-L1-X-L2-V: FVIII-L3-F2;

0⁾ F-L1-X-L2-V:F2-L3-FVIII;

(k) V-L2-X-L 1 -F 1 -L4-FVIII-L3 -F2;

(1) F2-L3-FVIII-L4-F 1 -L 1 -X-L2-V;

(m) FVIII-L2-F2-L4-V-L2-X-L1-F1; or

(n) F 1 -L 1 -X-L2-V-L4-F2-L2-F VIII,

wherein V comprises a VWF fragment,

each of LI and L3 comprises an optional linker,

L2 comprises an optional linker, e.g., a cleavable linker,

L4 is an optional linker, e.g., a processable linker,

FVIII comprises a FVIII protein,

X comprises one or more XTEN sequences,

Fl comprises an optional Ig constant region or a portion thereof,

F2 comprises an optional additional Ig constant region or a portion thereof;

(-) is a peptide bond or one or more amino acids; and

(:) is a chemical association or a physical association.

[0112] In some embodiments, the FVIII protein in any constructs or formulas disclosed herein can further comprises at least one, at least two, at least three, at least four, at least five, or at least six XTEN sequences, each of the XTEN sequences inserted immediately downstream of one or more amino acids in the FVIII protein or linked to the N-terminus or the C-terminus of the FVIII protein. Non-limiting examples of the XTEN insertion sites are disclosed elsewhere herein.

[0113] In one embodiment, (:) represents a chemical association, e.g., at least one non-peptide bond. In another embodiment, the chemical association, i.e., (:) is a covalent bond. In other embodiments, the chemical association, i.e., (:) is a non- covalent interaction, e.g., an ionic interaction, a hydrophobic interaction, a hydrophilic interaction, a Van der Waals interaction, or a hydrogen bond. In other embodiments, (:) is a non-peptide covalent bond. In still other embodiments, (:) is a peptide bond. In yet other embodiments, (:) represents a physical association between two sequences, wherein a portion of a first sequence is in close proximity to a second sequence such that the first sequence shields or blocks a portion of the second sequence from interacting with another moiety, and further that this physical association is maintained without allowing the second sequence to interact with other moieties. The orientation of the polypeptide formulas herein is listed from N-terminus (left) to C-terminus (right). For example, formula (n) Fl- L1-X-L2-V-L4-F2-L2-FVIII means formula NH2-F1 -LI -X-L2-V-L4-F2-L2- FVIII -COOH. In one embodiment, the formulas described herein can comprise any additional sequences between the two moieties. In another embodiment, the hyphen (-) indicates a peptide bond.

In one embodiment, either or both of the Ig constant region or a portion thereof (sometimes indicated herein by "F" or "Fl") and the additional Ig constant region or a portion thereof (sometimes indicated herein by "F2") linked to the VWF fragment or the FVIII protein can extend the half-life of the VWF fragment, the FVIII protein, or both. In another embodiment, a pair of the Ig constant region or a portion thereof (sometimes indicated herein by "F" or "Fl") and the additional Ig constant region or a portion thereof (sometimes indicated herein by "F2"), each of which are linked to the VWF fragment and the FVIII protein, provides a bond stronger than the non-covalent bond between the FVIII protein and the VWF fragment, i.e., a covalent bond, e.g., a disulfide bond, thereby preventing endogenous VWF from replacing the VWF fragment in vivo. Fl or F2 can comprise an Fc region or an FcRn binding partner. In other embodiments, either or both of Fl and F2 linked to the VWF fragment and/or the FVIII protein form a covalent bond {e.g., a disulfide bond) between Fl and F2, thereby placing the VWF fragment and the FVIII protein in close proximity to prevent interaction of the FVIII protein with the VWF fragment. In some embodiments, Fl and F2 are identical or different. Non-limiting examples of Fl and F2 can be selected from the group consisting of a CHI domain, a CH2 domain, a CH3 domain, a CH4 domain, a hinge domain, any functional fragments, derivatives, or analogs thereof, and two or more combinations thereof. In one embodiment, Fl, F2, or both comprise at least one CHI domain, at least one CH2 domain, at least one CH3 domain, at least one CH4 domain, or the functional fragments, derivatives, or analogs thereof. In another embodiment, Fl, F2, or both comprise at least one hinge domain or portion thereof and at least one CH2 domain or portion thereof (e.g., in the hinge-CH2 orientation). In other embodiments, Fl, F2, or both comprise at least one CH2 domain or portion thereof and at least one CH3 domain or portion thereof (e.g., in the CH2-CH3 orientation.) Examples of the

combination include, but are not limited to, a CH2 domain, a CH3 domain, and a hinge domain, which are also known as an Fc region (or Fc domain), e.g., a first Fc region or a first FcRn binding partner for Fl and a second Fc region or a second FcRn binding partner for F2. In other embodiments, Fl is linked to the VWF fragment by a linker, and/or F2 is linked to the FVIII protein by a linker. In some embodiments, Fl and/or F2 comprises, consisting essentially of, or consisting of a hinge region. Additional non-limiting examples of the Fc regions or the FcRn binding partners are described elsewhere herein.

In certain embodiments, a chimeric protein of the invention comprises two polypeptide chains, a first polypeptide chain comprising, consisting essentially of, or consisting of a VWF fragment comprising a D' domain and a D3 domain, an XTEN sequence, a first Ig constant region or a portion thereof (e.g., a first Fc region), and an optional linker between the VWF fragment and the XTEN sequence or the XTEN sequence or the first Ig constant region or a portion thereof and a second polypeptide chain comprising, consisting essentially of, or consisting of a FVIII protein and a second Ig constant region or a portion thereof (e.g., a second Fc region). The linker between the VWF fragment and the first Ig constant region or a portion thereof can be a cleavable linker, e.g., a thrombin cleavable linker, which can be cleaved at the site of coagulation. In some embodiments, the first polypeptide chain and the second polypeptide chain are associated with each other. The association between the first chain and the second chain prevents replacement of the first chain comprising the VWF fragment with endogenous VWF in vivo. In one embodiment, the association between the first chain and the second chain can be a covalent bond. In a particular embodiment, the covalent bond is a disulfide bond. In some embodiments, the FVIII protein in the second chain further comprises one or more XTEN sequences linked to the C-terminus or N-terminus of the FVIII protein or inserted immediately downstream of one or more amino acids (e.g., at least one insertion site disclosed herein) in the FVIII protein. Non-limiting examples of the insertion sites are described elsewhere herein.

[0116] In other embodiments, a chimeric protein of the invention comprises three polypeptide chains, wherein a first polypeptide chain comprises, consists essentially of, or consists of a heavy chain of a FVIII protein, a second polypeptide chain comprises, consists essentially of, or consists of a light chain of a FVIII protein fused to a first Ig constant region or a portion thereof (e.g., a first Fc region), and a third polypeptide chain comprises, consists essentially of, or consists of a VWF fragment comprising a D' domain and a D3 domain, an XTEN sequence, a second Ig constant region or a portion thereof (e.g., a second Fc region), and an optional linker between the XTEN sequence and the second Ig constant region or a portion thereof or the VWF fragment and the XTEN sequence. The linker in the third chain can be a cleavable linker, which is cleaved at the site of coagulation, e.g., a thrombin cleavage site. In some embodiments, the heavy chain FVIII or the light chain FVIII is linked to one or more XTEN sequences, which can be linked to the N-terminus, the C-terminus, or inserted within one or more insertion sites within the FVIII sequence. Non-limiting examples of the insertion sites are disclosed elsewhere herein.

[0117] In yet other embodiments, a chimeric protein of the invention comprises two polypeptide chains, a first polypeptide chain comprising, consisting essentially of, or consisting of a heavy chain of a FVIII protein and a second polypeptide chain comprising, consisting essentially of, or consisting of a light chain of a FVIII protein, a first Ig constant region or a portion thereof (e.g., a first Fc region), a first linker (e.g., a processable linker, which contains one or more protease cleavage sites comprising one or more intracellular processing sites), a VWF fragment, a second linker (e.g., a thrombin cleavable linker), an XTEN sequence, and a second Ig constant region or a portion thereof (e.g., a second Fc region), wherein the light chain of the FVIII protein is linked to the first Ig constant region or a portion thereof (e.g., the first Fc region), which is further linked to the VWF fragment by the first linker, and wherein the VWF fragment is linked to the XTEN sequence, which is further linked to the second Ig constant region or a portion thereof by the second linker. In certain embodiments, the first linker is a processable linker, and the second linker is a cleavable linker. Upon expression, the chimeric protein can be processed by an intracellular processing enzyme, which cleaves the processable linker, and thus the chimeric protein can comprise, consists essentially of, or consists of three polypeptide chains. In addition, the VWF fragment can be cleaved off at the site of coagulation due to the cleavable linker.

[0118] In certain embodiments, a chimeric protein of the invention comprises one polypeptide chain, which comprises a single chain FVIII protein, a first Ig constant region or a portion thereof (e.g., a first Fc region), a first linker (e.g., a processable linker), a VWF fragment, an XTEN sequence, a second linker (e.g., a thrombin cleavable linker), and a second Ig constant region or a portion thereof (e.g., a second Fc region), wherein the single chain FVIII protein is linked to the first Ig constant region or a portion thereof, which is also linked to the VWF fragment by the first linker, and the VWF fragment is linked to the XTEN sequence, which is further linked to the second Ig constant region or a portion thereof. In one embodiment, the VWF fragment and the XTEN sequence are linked by the second linker. In another embodiment, the XTEN sequence and the second Ig constant region or a portion thereof are linked by the second linker. In other embodiments, the second chain further comprises a third linker. The single polypeptide chain can thus comprise the VWF fragment linked to the XTEN sequence by the second linker and the XTEN linked to the second Ig constant region or a portion thereof by the third linker. The second linker and the third linker can be identical or different. In one embodiment, the first linker is a processable linker. In another embodiment, the second linker or the third linker is a cleavable linker comprising one or two cleavable sites. In a specific

embodiment, the second linker is a thrombin cleavable linker. The linkers useful in the invention are described elsewhere herein.

(2) FVIII, XTEN, and Fc

[0119] A chimeric protein of the invention also comprises (i) a FVIII protein, (ii) an XTEN sequence (a first half-life extender), and (iii) an Ig constant region or a portion thereof (a second half-life extender), in which the XTEN sequence is linked to the FVIII protein by an optional linker and the Ig constant region or a portion thereof by an additional optional linker. The XTEN sequence and the Ig constant region or a portion thereof can be used together to extend half-life of the FVIII protein. In one embodiment, the chimeric protein is a monomer. In another embodiment, the chimeric protein is a dimer (a homodimer or a heterodimer).

[0120] The present invention is also directed to a chimeric protein comprising (i) a

FVIII protein, (ii) an XTEN sequence, (iii) an Ig constant region or a portion thereof {i.e., a first Ig constant region or a portion thereof, "F," or "Fl "), and (iv) an additional Ig constant region or a portion thereof {i.e., a second Ig constant region or a portion thereof or "F2"). In one embodiment, the XTEN sequence is linked to the FVIII protein at the C-terminus or the N-terminus or inserted immediately downstream of one or more amino acids in the FVIII protein {e.g., one or more XTEN insertion sites), the FVIII protein is linked to the first Ig constant region or a portion thereof, and the first Ig constant region or a portion thereof and the second Ig constant region or a portion thereof are associated with or linked to each other by an optional linker. In certain aspects, the chimeric protein is a monomer-dimer hybrid, which comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a FVIII protein, an XTEN sequence, and a first Ig constant region or a portion thereof, and the second polypeptide chain comprises, consists essentially of, or consists of a second Ig constant region or a portion thereof without the FVIII protein and wherein the first chain and the second chain are associated with each other. The association between the Ig constant region or a portion thereof {e.g., the first Fc region) and the additional Ig constant region or a portion thereof {e.g., a second Fc region) is a chemical association or a physical association. In certain

embodiments, the chemical association is a covalent bond. In other embodiments, the chemical association is a non-covalent interaction, e.g., an ionic interaction, a hydrophobic interaction, a hydrophilic interaction, a Van der Waals interaction, or a hydrogen bond. In other embodiments, the association is a non-peptide covalent bond. In still other embodiments, the association is a peptide bond.

[0121] In other aspects, the chimeric protein is a single polypeptide chain

comprising a FVIII protein, an XTEN sequence, a first Ig constant region or a portion thereof, a linker, e.g., a processable linker, and a second Ig constant region or a portion thereof, wherein the single polypeptide chain is processed after expression by an intracellular enzyme and becomes two polypeptide chains.

[0122] In one embodiment, the Ig constant region or a portion thereof (sometimes indicated herein by "F" or "Fl ") linked to the FVIII protein can extend the half-life of the FVIII protein together with the XTEN sequence. In another embodiment, the Ig constant region or a portion thereof ("F" or "Fl") is an Fc region or an FcRn binding partner described elsewhere herein.

[0123] In other embodiments, the additional Ig constant region or a portion

thereof (sometimes indicated herein by "F2" or a second Ig constant region or a portion thereof) associated with or linked to the first Ig constant region or a portion thereof can also extend the half-life of the FVIII protein. In other embodiments, the second Ig constant region or a portion thereof ("F2") together with the first Ig constant region or a portion thereof and the XTEN sequence can extend the half-life of the FVIII protein. The additional Ig constant region or a portion thereof can be an Fc region or an FcRn binding partner described elsewhere herein.

[0124] In certain embodiments, the second Ig constant region or a portion thereof associated with the first Ig constant region or a portion thereof is further linked to a VWF fragment described elsewhere herein and an optional XTEN sequence.

[0125] In some embodiments, either or both of the Ig constant region or a portion thereof ("F" or "Fl" or a first Ig constant region or a portion thereof) and an additional Ig constant region or a portion thereof (i.e., a second Ig constant region or a portion thereof or "F2") (indicated in this paragraph as "the Ig constant regions or portion thereof) can include, but not limited to, a CHI domain, a CH2 domain, a CH3 domain, a CH4 domain, a hinge domain, any functional fragments, derivatives, or analogs thereof or two or more combinations thereof. In one embodiment, the Ig constant region or a portion thereof comprises at least one CHI domain, at least one CH2 domain, at least one CH3 domain, at least one CH4 domain, or the functional fragments, derivatives, or analogues thereof. In another embodiment, the Ig constant region or a portion thereof comprises at least one hinge domain or portion thereof and at least one CH2 domain or portion thereof (e.g., in the hinge-CH2 orientation). In other embodiments, the Ig constant domain or portion thereof comprises at least one CH2 domain or portion thereof and at least one CH3 domain or portion thereof (e.g., in the CF2-CH3 orientation). Examples of the combination include, but are not limited to, a CH2 domain, a CH3 domain, and a hinge domain, which are also known as an Fc region (or Fc domain), e.g., first Fc region. Additional examples of the Ig constant regions or portion thereof are described elsewhere herein.

[0126] The chimeric protein of the invention can have an extended half-life of the

FVIII protein compared to wild-type FVIII. In one embodiment, the half-life of the FVIII protein is extended at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, or at least about 12 times longer than the half-life of wild type FVIII. In another embodiment, the half-life of the FVIII protein is at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours.

(3) FVIII, XTEN, and VWF

[0127] In one aspect, a chimeric protein of the present invention comprises (i) a

FVIII protein, (ii) an XTEN sequence, and (iii) a VWF fragment comprising a D' domain and a D3 domain of VWF, wherein the FVIII protein is linked to the XTEN sequence and wherein the FVIII protein is associated with or linked to the VWF fragment. In one embodiment, the VWF fragment of the chimeric protein described herein is not capable of binding to a VWF clearance receptor. In another embodiment, the VWF fragment is capable of protecting the FVIII protein from one or more protease cleavages, protecting the FVIII protein from activation, stabilizing the heavy chain and/or the light chain of the FVIII protein, or preventing clearance of the FVIII protein by one or more scavenger receptors. In other embodiments, the VWF fragment prevents or inhibits binding of endogenous VWF to the VWF binding site in the FVIII protein. The VWF binding site can be located in the A3 domain or the C2 domain of the FVIII protein or both the A3 domain and the C2 domain. In a specific embodiment, the VWF binding site comprises the amino acid sequence corresponding to amino acids 1669 to 1689 and/or amino acids 2303 to 2332 of SEQ ID NO: 2.

[0128] In another aspect, a chimeric protein comprises (i) a FVIII protein, (ii) an

XTEN sequence, (iii) a VWF fragment, which comprises a D' domain and a D3 domain of VWF, and (iv) an Ig constant region or a portion thereof, wherein the XTEN sequence is linked to the FVIII protein at the C-terminus or the N-terminus or inserted immediately downstream of one or more amino acids (e.g., one or more XTEN insertion sites disclosed herein) in the FVIII protein, the VWF fragment is linked to or associated with the FVIII protein or the XTEN sequence, and the Ig constant region or a portion thereof is linked to the FVIII protein, the XTEN sequence, the VWF fragment, or any combinations thereof. The Ig constant region or a portion thereof useful for chimeric proteins of the invention is described elsewhere herein. In one embodiment, the Ig constant region or a portion thereof is capable of extending the half-life of a FVIII protein. In another embodiment, the Ig constant region or a portion thereof comprises a first Fc region or a first FcRn binding partner. In yet other embodiments, the Ig constant region or a portion thereof is linked to the FVIII protein by an optional linker. In still other embodiments, the linker comprises a cleavable linker. The chimeric protein can be a single polypeptide chain, i.e., a monomer (i.e., a single chain), containing

(i) , (ii), (iii), and (iv) or two chains containing a first chain comprising (i) and (ii) and a second chain comprising (iii) and (iv). In other aspects, the chimeric protein is a dimer (e.g., a homodimer or a heterodimer). In one embodiment, the chimeric protein comprises two chains, each comprising (i), (ii), (iii), and (iv).

[0129] In certain embodiments, a chimeric protein comprises (i) a FVIII protein,

(ii) an XTEN sequence, (iii) a VWF fragment, which comprises a D' domain and a D3 domain of VWF, (iv) an Ig constant region or a portion thereof (sometimes also indicated as "F," "a first Ig constant region or a portion thereof, or "F2"), and (v) an additional Ig constant region or a portion thereof (sometimes also indicated as "F2" or "a second Ig constant region or a portion thereof), wherein (1) the FVIII protein is linked to the XTEN sequence at the C-terminus or N-terminus of the FVIII protein or inserted immediately downstream of one or more amino acids (e.g., one or more XTEN insertion sites disclosed herein) in the FVIII protein, (2) either the XTEN sequence or the FVIII protein is linked to the Ig constant region or a portion thereof, (3) the VWF fragment is linked to the second Ig constant region or a portion thereof, and (4) the Ig constant region or a portion thereof is associated with the second Ig constant region or a portion thereof. In one embodiment, the Ig constant region or a portion thereof linked to the FVII protein or the XTEN sequence is further linked to the VWF fragment by a linker, e.g., a processable linker. In another embodiment, the additional Ig constant region or a portion thereof useful for chimeric proteins of the invention can further be linked to the FVIII protein or the Ig constant region or a portion thereof by an optional linker, e.g., a processable linker. In some embodiments, a pair of the Ig constant region or a portion thereof and the additional Ig constant region or a portion thereof, each of which are linked to the VWF fragment and the FVIII protein, provides a bond stronger than the non-covalent bond between the FVIII protein and the VWF fragment, i.e., a covalent bond, e.g., a disulfide bond, thereby preventing endogenous VWF from replacing the VWF fragment in vivo. In other embodiments, either or both of the Ig constant region or a portion thereof and the additional Ig constant region or a portion thereof are capable of extending a half- life of the FVIII protein or the VWF fragment. In other embodiments, the additional Ig constant region or a portion thereof comprises a second Fc region or an FcRn binding partner. The Ig constant region or a portion thereof and the additional Ig constant region or a portion thereof in the chimeric proteins are identical or different.

In certain embodiments, the Ig constant region or a portion thereof and the additional Ig constant region or a portion thereof are associated by a chemical association or a physical association. In one embodiment, the chemical association, i.e., (:), is at least one non-peptide bond. In certain embodiments, the chemical association, i.e., (:), is a covalent bond. In other embodiments, the chemical association, i.e., (:), is a non-covalent interaction, e.g., an ionic interaction, a hydrophobic interaction, a hydrophilic interaction, a Van der Waals interaction, or a hydrogen bond. In other embodiments, (:) is a non-peptide covalent bond. In still other embodiments, (:) is a peptide bond. In yet other embodiments, (:) represents a physical association between two sequences, wherein a portion of a first sequence is in close proximity to a second sequence such that the first sequence shields or blocks a portion of the second sequence from interacting with another moiety. In some embodiments, the association between the Ig constant region or a portion thereof and the additional Ig constant region or a portion thereof can be a covalent bond, e.g., a disulfide bond, which prevents replacement the VWF fragment or the polypeptide containing the VWF fragment with endogenous VWF. Therefore, preventing interaction between the FVIII protein and endogenous VWF reduces or eliminates this half-life limiting factor for the FVIII protein, and thus the half-life of the FVIII protein is extended compared to a FVIII protein without the VWF protein or wild-type FVIII.

In other aspects, a chimeric protein comprises a formula comprising

(1) FVIII(X1)-L1-F1 :V-L2-X2-L3-F2;

(2) FVIII(X1)-L1-F1 :F2-L3-X2-L2-V;

(3) F 1 -L 1 -FVIII(X 1 ) : V-L2-X2-L3 -F2;

(4) F 1 -L 1 -FVIII(X 1 ) :F2-L3 -X2-L2-V;

(5) FVIII(X1 )-L 1 -F 1 -L4-V-L2-X2-L3-F2;

(6) FVIII(X1 )-L 1 -F 1 -L4-F2-L3-X2-L2-V;

(?) F 1 -L 1 -FVIII(X1 )-L4-V-L2-X2-L3-F2, or

(8) F 1 -L 1 -FVIII(X1 )-L4- F2-L3-X2-L2-V, wherein FVIII(Xl) comprises a FVIII protein and one or more XTEN sequences, wherein the one or more XTEN sequence are linked to the N-terminus or C- terminus of the FVIII protein or inserted immediately downstream of one or more amino acids {e.g., one or more XTEN insertion sites disclosed herein) in the FVIII protein; each of LI, L2, or L3 comprises an optional linker, e.g., a cleavable linker;

L4 is a linker, e.g., a processable linker;

X2 comprises one or more optional XTEN sequences;

Fl comprises an Ig constant region or a portion thereof;

F2 comprises an optional additional Ig constant region or a portion thereof, and V comprises a VWF fragment; (-) is a peptide bond or one or more amino acids; and

(:) comprises a chemical association or a physical association. In one embodiment, (:) represents a chemical association, e.g., at least one non-peptide bond. In another embodiment, the chemical association, i.e., (:) is a covalent bond. In other embodiments, the chemical association, i.e., (:) is a non-covalent interaction, e.g., an ionic interaction, a hydrophobic interaction, a hydrophilic interaction, a Van der Waals interaction, or a hydrogen bond. In other embodiments, (:) is a non- peptide covalent bond. In still other embodiments, (:) is a peptide bond. In yet other embodiments, (:) represents a physical association between two sequences, wherein a portion of a first sequence is in close proximity to a second sequence such that the first sequence shields or blocks a portion of the second sequence from interacting with another moiety, and further that this physical association is maintained without allowing the second sequence to interact with other moieties. The orientation of the polypeptide formulas herein is listed from N-terminus (left) to C-terminus (right). For example, formula V-X-FVIII means formula NH2-V- X-FVIII -COOH. In one embodiment, the formulas described herein can comprise any additional sequences between the two moieties. For example, formula V-X- FVIII can further comprise any sequences at the N-terminus of V between V and X, between X and FVIII, or at the C-terminus of FVIII unless otherwise specified. In another embodiment, the hyphen (-) indicates a peptide bond. In one aspect, the chimeric protein comprises two polypeptide chains, (A) a first chain comprising (i) a single chain FVIII protein (ii) an XTEN sequence, and (iii) a first Ig constant region or a portion thereof, e.g., a first Fc region or FcRn binding partner, wherein the XTEN sequence is linked to the FVIII protein at the N-terminus or C-terminus or inserted immediately downstream of one or more amino acids of the FVIII protein {e.g., one or more XTEN insertion sites disclosed herein) and the first Ig constant region or a portion thereof is linked to the XTEN sequence when the XTEN sequence is linked to the FVIII protein at the N-terminus or the C-terminus or the FVIII protein when the XTEN sequence is inserted within the FVIII protein, and (B) a second chain comprising (iv) a VWF fragment comprising a D' domain and a D3 domain, (v) a linker, and (vi) a second Ig constant region or a portion thereof, e.g., a second Fc region or a second FcRn binding partner, wherein the VWF fragment is linked to the linker, e.g., a cleavable linker, which is further linked to the second Ig constant region or a portion thereof, and wherein the first polypeptide chain and the second

polypeptide chain are associated with each other, e.g., a covalent bond, e.g., a disulfide bond. In one embodiment, the linker is a cleavable linker described elsewhere herein, e.g., a thrombin cleavable linker. In some embodiments, the second chain comprises one or more XTEN sequences between (iv) and (v) or (v) and (vi).

[0133] In other aspects, the chimeric protein comprises one polypeptide chain comprising (i) a single chain FVIII protein (ii) an XTEN sequence, (iii) a first Ig constant region or a portion thereof, e.g., a first Fc region or a first FcRn binding partner, (iv) a first linker, (v) a VWF fragment comprising a D' domain and a D3 domain, (vi) a second linker, and (vii) a second Ig constant region or a portion thereof, e.g., a second Fc region or a second FcRn binding partner, wherein (i) to (vii) are linked in the order or in any orders. In one embodiment, the first linker is a processable linker, which can be intracellularly processed or cleaved after expression and makes the single polypeptide chain into two polypeptide chains. In another embodiment, the second linker is a cleavable linker described herein, e.g., a thrombin cleavable linker. The XTEN sequence used herein can be linked to the FVIII protein by an optional linker at the N-terminus or the C terminus of the FVIII protein or inserted immediately downstream of one or more amino acids {e.g., one or more XTEN insertion sites) in the FVIII protein.

[0134] In certain aspects, a chimeric protein comprises three polypeptide chains,

(A) a first polypeptide chain comprising (i) a heavy chain of a FVIII protein and (ii) an XTEN sequence, which are linked to each other and (B) a second polypeptide chain comprising (iii) a light chain of the FVIII protein and (iv) a first Ig constant region or a portion thereof, e.g., a first Fc region or a first FcRn binding partner, which are linked to each other, and (C) a third polypeptide chain comprising (v) a VWF fragment comprising a D' domain and a D3 domain, (vi) a linker, and (vii) a second Ig constant region or a portion thereof, e.g., a second Fc region or a second FcRn binding partner, wherein the second chain is associated with the first chain and the third chain. In one embodiment, the association between the first chain and the second chain is a chemical association or a physical association. For example, the association between the first chain and the second chain can be a metal bond. In another embodiment, the association between the second chain and the third chain is also a chemical association or a physical association, e.g., a covalent bond or a non-covalent bond. In certain embodiments, the association between the second chain and the third chain is through the two Ig constant regions or a portion thereof and is a disulfide bond. The bonding between the second chain and the third chain prevents or inhibits binding of the FVIII protein with endogenous VWF, thus preventing the FVIII protein being cleared by the VWF clearance pathway. In some embodiments, the linker is a processable linker, which is intracellularly cleaved after expression in a host cell. The XTEN sequence used herein is linked to the FVIII protein by an optional linker at the N-terminus or C terminus of the FVIII protein or inserted immediately downstream of one or more amino acids {e.g., one or more XTEN insertion sites) in the FVIII protein.

[0135] In certain embodiments, the VWF fragment is directly linked to the FVIII protein, which comprises one or more XTENs, by a peptide bond or a linker. As one way of linking the VWF fragment and the FVIII protein, in which one or more XTENs are inserted or linked, through a direct link {e.g. a peptide bond) or a linker, an enzymatic ligation {e.g., sortase) can be employed. For example, sortase refers to a group of prokaryotic enzymes that modify surface proteins by recognizing and cleaving a carboxyl-terminal sorting signal. For most substrates of sortase enzymes, the recognition signal consists of the motif LPXTG (Leu-Pro- any-Thr-Gly (SEQ ID NO: 51), then a highly hydrophobic transmembrane sequence, then a cluster of basic residues such as arginine. Cleavage occurs between the Thr and Gly, with transient attachment through the Thr residue to the active site Cys residue of a ligation partner, followed by transpeptidation that attaches the protein covalently to the cell wall. In some embodiments, the ligation partner contains Gly(n). In other embodiments, the chimeric protein further comprises a sortase recognition motif. In some embodiments, the VWF fragment is attached to FVIII comprising one or more XTENs inserted within or linked to using sortase mediated in vitro protein ligation.

[0136] In one embodiment, a VWF fragment linked to a sortase recognition motif by an optional linker can be fused to a FVIII protein linked to Gly(n) by a sortase, wherein n can be any integer and wherein one or more XTENs are inserted within or linked to the FVIII protein. A ligation construct comprises the VWF fragment (N-terminal portion of the construct) and the FVIII protein, in which one or more XTENs are inserted or linked (C-terminal portion of the construct), wherein the sortase recognition motif is inserted in between. Another ligation construct comprises the VWF fragment (N-terminal portion of the construct, the linker, the sortase recognition motif, and the FVIII protein, in which one or more XTENs are inserted or linked (C-terminal portion of the construct). In another embodiment, a FVIII protein linked to a sortase recognition motif by an optional linker can be fused to a VWF fragment linked to Gly(n) by a sortase, wherein n is any integer. A resulting ligation construct comprises the FVIII protein (N-terminal portion of the construct), in which one or more XTENs are inserted or linked, and the VWF fragment (C-terminal portion of the construct), wherein the sortase recognition motif is inserted in between. Another resulting ligation construct comprises the FVIII protein (N-terminal portion of the construct), in which one or more XTENs are inserted or linked, the linker, the sortase recognition motif, and the VWF fragment (C-terminal portion of the construct). In other embodiments, a VWF fragment linked to a sortase recognition motif by a first optional linker can be fused to a heterologous moiety, e.g., an immunoglobulin constant region or a portion thereof, e.g., an Fc region, linked to a thrombin cleavage site by a second optional linker. A resulting construct can comprise the VWF fragment (N- terminal portion), the first linker, the sortase recognition motif, the protease cleavage site, the second optional linker, and the heterologous moiety.

In some embodiments, the VWF fragment is associated with the FVIII protein. The association between the VWF fragment and the FVIII protein can be a chemical association or a physical association. The chemical association can be a non-covalent interaction, e.g., an ionic interaction, a hydrophobic interaction, a hydrophilic interaction, a Van der Waals interaction, or a hydrogen bond. In yet other embodiments, the association between the FVIII protein and the VWF fragment is a physical association between two sequences, e.g., due to an additional association between the sequence having the FVIII protein and the sequence having the VWF fragment, wherein a portion of a first sequence is in close proximity to a second sequence such that the first sequence shields or blocks a portion of the second sequence from interacting with another moiety. [0138] As a result of preventing or inhibiting endogenous VWF interaction with the FVIII protein by the VWF fragment, the chimeric protein described herein have an extended half-life compared to wild-type FVIII or the corresponding chimeric protein without the VWF fragment. In one embodiment, the half-life of the FVIII protein is extended at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, or at least about 12 times longer than a FVIII protein without the VWF fragment. In another embodiment, the half-life of the FVIII protein is at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours. In a particular embodiment, the half-life of the FVIII protein is extended at least 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, or at least about 27 hours in HemA mice.

A) Von Willebrand Factor (VWF) Fragments

[0139] VWF (also known as F8VWF) is a large multimeric glycoprotein present in blood plasma and produced constitutively in endothelium (in the Weibel-Palade bodies), megakaryocytes (a-granules of platelets), and subendothelian connective tissue. The basic VWF monomer is a 2813 amino acid protein. Every monomer contains a number of specific domains with a specific function, the D'/D3 domain (which binds to Factor VIII), the Al domain (which binds to platelet GPIb- receptor, heparin, and/or possibly collagen), the A3 domain (which binds to collagen), the CI domain (in which the RGD domain binds to platelet integrin llbp3 when this is activated), and the "cysteine knot" domain at the C-terminal end of the protein (which VWF shares with platelet-derived growth factor

(PDGF), transforming growth factor-β (TGFP) and β-human chorionic

gonadotropin (PHCG).

[0140] The term "a VWF fragment" as used herein includes, but is not limited to, functional VWF fragments comprising a D' domain and a D3 domain, which are capable of inhibiting binding of endogenous VWF to FVIII. In one embodiment, the VWF fragment binds to the FVIII protein. In another embodiment, the VWF fragment blocks the VWF binding site on the FVIII protein, thereby inhibiting interaction of the FVIII protein with endogenous VWF. The VWF fragments include derivatives, variants, mutants, or analogues that retain these activities of VWF.

[0141] The 2813 monomer amino acid sequence for human VWF is reported as

Accession Number NP 000543.2 in Genbank. The nucleotide sequence encoding the human VWF is reported as Accession Number NM_000552.3_ in

Genbank. The nucleotide sequence of human VWF is designated as SEQ ID NO: 1. SEQ ID NO: 2 is the amino acid sequence encoded by SEQ ID NO: 1. Each domain of VWF is listed in Table 1.

TABLE 1. VWF Sequences

VWF domains Amino acid Sequence

VWF Signal Peptide MI PARFAGVL LALALILPGT LC

22

(Amino acids 1 to 22

of SEQ ID NO: 2)

VWF D1D2 region 2 3 AEGTRGRS

STARCSLFGS DFV TFDGSM

(Amino acids 23 to 5 YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK 763 of SEQ ID NO: 2) RVSLSVYLGE FFDIHLFV G

1 01 TVTQGDQRVS MPYASKGLYL ETEAGYYKLS

GEAYGFVARI DGSGNFQVLL

1 1 SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL

TSDPYDFANS WALSSGEQWC

2 01 ERASPPSSSC NISSGEMQKG LWEQCQLLKS

TSVFARCHPL VDPEPFVALC

2 51 EKTLCECAGG LECACPALLE YARTCAQEGM

VLYGWTDHSA CSPVCPAGME

3 01 YRQCVSPCAR TCQSLHINEM CQERCVDGCS

CPEGQLLDEG LCVESTECPC

3 51 VHSGKRYPPG TSLSRDCNTC ICRNSQWICS

NEECPGECLV TGQSHFKSFD

4 01 NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC TRSVTVRLPG

45 LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL

RIQHTVTASV RLSYGEDLQM

50 DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN

QGDDFLTPSG LAEPRVEDFG

55 NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS

EEACAVLTSP TFEACHRAVS

60 PLPYLRNCRY DVCSCSDGRE CLCGALASYA

AACAGRGVRV AWREPGRCEL

65 NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN

EACLEGCFCP PGLYMDERGD

70 CVPKAQCPCY YDGEIFQPED IFSDHHTMCY

CEDGFMHCTM SGVPGSLLPD AVLSSPLSHR SKR

76

VWF D' Domain 764 SLSCRPP MVKLVCPADN

LRAEGLECTK

TCONYDLECM

!0: SMGCVSGCLC PPGMVRHENR CVALERCPCF

HQGKEYAPGE TVKIGCNTCV CRDRKWNCTD HVCDAT

VWF D3 Domain 867 CSTI GMAHYLTFDG

LKYLFPGECQ YVLVQDYCGS

90 1 NPGTFRILVG NKGCSHPSVK CKKRV ILVE

GGEIELFDGE VNVKRPMKDE

95 1 THFEVVESGR YIILLLGKAL SVVWDRHLSI

SVVLKQTYQE KVCGLCGNFD

10 01 GIQNNDLTSS NLQVEEDPVD FGNSWKVSSQ

CADTRKVPLD SSPATCHNNI

10 51 MKQTMVDSSC RILTSDVFQD CNKLVDPEPY

LDVCIYDTCS CESIGDCACF

11 01 CD IAAYAHV CAQHGKVVTW RTATLCPQSC

EERNLRENGY ECEWRYNSCA

1 51 PACQVTCQHP EPLACPVQCV EGCHAHCPPG

KILDELLQTC VDPEDCPVCE

1201 KVTLNPSDPE HCQICHCDVV

NLTCEACQEP

^■:4 o

VWF Al Domain 1241 GGLVVPPTE

1251 PVSPTTLYVE DISEPPLHDF YCSRLLDLVF

LLDGSSRLSE AEFEVLKAFV VDMMERLRIS QKWVRVAVVE YHDGSHAYIG LKDRKRPSEL RRIASQVKYA

135: GSQVASTSEV LKYTLFQIFS KIDRPEASRI

ALLLMASQEP QRMSRNFVRY

140: VQGLKKKKVI VIPVGIGPHA NLKQIRLIEK QAPENKAFVL SSVDELEQQR

DEIVSYLCDL APEAPPPTLP PDMAQVTVG

1479

P

GLLGVSTLGP KRNSMVLDVA FVLEGSDKIG EADFNRSKEF MEEVIQRMDV GQDSIHVTVL QYSYMVTVEY PFSEAQSKGD ILQRVREIRY QGGNRTNTGL ALRYLSDHSF LVSQGDREQA

1600

PNLVYMVTGN PASDEIKRLP GDIQVVPIGV GPNANVQELE RIGWPNAPIL IQDFETLPRE APDLVLQRCC SGEGLQIPTL SPAPDCSQPL DVILLLDGSS SFPASYFDEM KSFAKAFISK ANIGPRLTQV SVLQYGSITT IDVPWNVVPE KAHLLSLVDV MQREGGPSQI GDALGFAVRY LTSEMHGARP GASKAVVILV TDVSVDSVDA AADAARSNRV TVFPIGIGDR YDAAQLRILA GPAGDSNVVK LQRIEDLPTM VTLGNSFLHK LCSGFVRICM DEDGNEKRPG DVWTLPDQCH TVTCQPDGQT LLKSHRVNCD RGLRPSCPNS QSPVKVEETC GCRWTCPCVC TGSSTRHIVT FDGQNFKLTG SCSYVLFQNK EQDLEVILHN GACSPGARQG CMKSIEVKHS ALSVEXHSDM EVTVNGRLVS VPYVGGNMEV NVYGAIMHEV RFNHLGHIFT FTPQNNEFQL QLSPKTFASK TYGLCGICDE NGANDFMLRD GTVTTDWKTL VQEWTVQRPG QTCQPILEEQ CLVPDSSHCQ VLLLPLFAEC HKVLAPATFY AICQQDSCHQ EQVCEVIASY AHLCRTNGVC VDWRTPDFCA MSCPPSLVYN HCEHGCPRHC DGNVSSCGDH PSEGCFCPPD KVMLEGSCVP EEACTQCIGE DGVQHQFLEA WVPDHQPCQI CTCLSGRKVN CTTQPCPTAK APTCGLCEVA RLRQNADQCC PEYECVCDPV SCDLPPVPHC ERGLQPTLTN PGECRPNFTC ACRKEECKRV SPPSCPPHRL PTLRKTQCCD EYECACNCVN STVSCPLGYL ASTATNDCGC TTTTCLPDKV CVHRSTIYPV GQFWEEGCDV CTCTDMEDAV MGLRVAQCSQ KPCEDSCRSG FTYVLHEGEC CGRCLPSACE VVTGSPRGDS QSSWKSVGSQ WASPENPCLI NECVRVKEEV FIQQRNVSCP QLEVPVCPSG FQLSCKTSAC CPSCRCERME ACMLNGTVIG PGKTVMIDVC 2601 TTCRCMVQVG VISGFKLECR KTTCNPCPLG

YKEENNTGEC CGRCLPTACT

2651 IQLRGGQIMT LKRDETLQDG CDTHFCKVNE

RGEYFWEKRV TGCPPFDEHK

2701 CLAEGGKIMK IPGTCCDTCE EPECNDITAR

LQYVKVGSCK SEVEVDIHYC

2751 QGKCASKAMY SIDINDVQDQ CSCCSPTRTE

PMQVALHCTN GSVVYHEVLN

2801 AMECKCSPRK CSK

Nucleotide Sequence (SEQ ID NO: 1)

Full-length VWF 1 ATGATTCCTG CCAGATTTGC

CGGGGTGCTG CTTGCTCTGG CCCTCATTTT

51 GCCAGGGACC CTTTGTGCAG

AAGGAACTCG CGGCAGGTCA TCCACGGCCC

101 GATGCAGCCT TTTCGGAAGT

GACTTCGTCA ACACCTTTGA TGGGAGCATG

151 TACAGCTTTG CGGGATACTG

CAGTTACCTC CTGGCAGGGG GCTGCCAGAA

201 ACGCTCCTTC TCGATTATTG

GGGACTTCCA GAATGGCAAG AGAGTGAGCC

251 TCTCCGTGTA TCTTGGGGAA

TTTTTTGACA TCCATTTGTT TGTCAATGGT

301 ACCGTGACAC AGGGGGACCA

AAGAGTCTCC ATGCCCTATG CCTCCAAAGG

351 GCTGTATCTA GAAACTGAGG

CTGGGTACTA CAAGCTGTCC GGTGAGGCCT

401 ATGGCTTTGT GGCCAGGATC

GATGGCAGCG GCAACTTTCA AGTCCTGCTG

451 TCAGACAGAT ACTTCAACAA

GACCTGCGGG CTGTGTGGCA ACTTTAACAT

501 CTTTGCTGAA GATGACTTTA

TGACCCAAGA AGGGACCTTG ACCTCGGACC

551 CTTATGACTT TGCCAACTCA

TGGGCTCTGA GCAGTGGAGA ACAGTGGTGT

601 GAACGGGCAT CTCCTCCCAG

CAGCTCATGC AACATCTCCT CTGGGGAAAT

651 GCAGAAGGGC CTGTGGGAGC

AGTGCCAGCT TCTGAAGAGC ACCTCGGTGT

701 TTGCCCGCTG CCACCCTCTG

GTGGACCCCG AGCCTTTTGT GGCCCTGTGT

751 GAGAAGACTT TGTGTGAGTG

TGCTGGGGGG CTGGAGTGCG CCTGCCCTGC

801 CCTCCTGGAG TACGCCCGGA

CCTGTGCCCA GGAGGGAATG GTGCTGTACG

851 GCTGGACCGA CCACAGCGCG

TGCAGCCCAG TGTGCCCTGC TGGTATGGAG

901 TATAGGCAGT GTGTGTCCCC

TTGCGCCAGG ACCTGCCAGA GCCTGCACAT

951 CAATGAAATG TGTCAGGAGC GATGCGTGGA TGGCTGCAGC TGCCCTGAGG

1001 GACAGCTCCT GGATGAAGGC

CTCTGCGTGG AGAGCACCGA GTGTCCCTGC

1051 GTGCATTCCG GAAAGCGCTA

CCCTCCCGGC ACCTCCCTCT CTCGAGACTG

1101 CAACACCTGC ATTTGCCGAA

ACAGCCAGTG GATCTGCAGC AATGAAGAAT

1151 GTCCAGGGGA GTGCCTTGTC

ACTGGTCAAT CCCACTTCAA GAGCTTTGAC

1201 AACAGATACT TCACCTTCAG

TGGGATCTGC CAGTACCTGC TGGCCCGGGA

1251 TTGCCAGGAC CACTCCTTCT

CCATTGTCAT TGAGACTGTC CAGTGTGCTG

1301 ATGACCGCGA CGCTGTGTGC

ACCCGCTCCG TCACCGTCCG GCTGCCTGGC

1351 CTGCACAACA GCCTTGTGAA

ACTGAAGCAT GGGGCAGGAG TTGCCATGGA

1401 TGGCCAGGAC ATCCAGCTCC

CCCTCCTGAA AGGTGACCTC CGCATCCAGC

1451 ATACAGTGAC GGCCTCCGTG

CGCCTCAGCT ACGGGGAGGA CCTGCAGATG

1501 GACTGGGATG GCCGCGGGAG

GCTGCTGGTG AAGCTGTCCC CCGTCTATGC

1551 CGGGAAGACC TGCGGCCTGT

GTGGGAATTA CAATGGCAAC CAGGGCGACG

1601 ACTTCCTTAC CCCCTCTGGG

CTGGCRGAGC CCCGGGTGGA GGACTTCGGG

1651 AACGCCTGGA AGCTGCACGG

GGACTGCCAG GACCTGCAGA AGCAGCACAG

1701 CGATCCCTGC GCCCTCAACC

CGCGCATGAC CAGGTTCTCC GAGGAGGCGT

1751 GCGCGGTCCT GACGTCCCCC

ACATTCGAGG CCTGCCATCG TGCCGTCAGC

1801 CCGCTGCCCT ACCTGCGGAA

CTGCCGCTAC GACGTGTGCT CCTGCTCGGA

1851 CGGCCGCGAG TGCCTGTGCG

GCGCCCTGGC CAGCTATGCC GCGGCCTGCG

1901 CGGGGAGAGG CGTGCGCGTC

GCGTGGCGCG AGCCAGGCCG CTGTGAGCTG

1951 AACTGCCCGA AAGGCCAGGT

GTACCTGCAG TGCGGGACCC CCTGCAACCT

2001 GACCTGCCGC TCTCTCTCTT

ACCCGGATGA GGAATGCAAT GAGGCCTGCC

2051 TGGAGGGCTG CTTCTGCCCC

CCAGGGCTCT ACATGGATGA GAGGGGGGAC

2101 TGCGTGCCCA AGGCCCAGTG

CCCCTGTTAC TATGACGGTG AGATCTTCCA

2151 GCCAGAAGAC ATCTTCTCAG

ACCATCACAC CATGTGCTAC TGTGAGGATG

2201 GCTTCATGCA CTGTACCATG

AGTGGAGTCC CCGGAAGCTT GCTGCCTGAC 2251 GCTGTCCTCA GCAGTCCCCT

GTCTCATCGC AGCAAAAGGA GCCTATCCTG

2301 TCGGCCCCCC ATGGTCAAGC

TGGTGTGTCC CGCTGACAAC CTGCGGGCTG

2351 AAGGGCTCGA GTGTACCAAA

ACGTGCCAGA ACTATGACCT GGAGTGCATG

2401 AGCATGGGCT GTGTCTCTGG

CTGCCTCTGC CCCCCGGGCA TGGTCCGGCA

2451 TGAGAACAGA TGTGTGGCCC

TGGAAAGGTG TCCCTGCTTC CATCAGGGCA

2501 AGGAGTATGC CCCTGGAGAA

ACAGTGAAGA TTGGCTGCAA CACTTGTGTC

2551 TGTCGGGACC GGAAGTGGAA

CTGCACAGAC CATGTGTGTG ATGCCACGTG

2601 CTCCACGATC GGCATGGCCC

ACTACCTCAC CTTCGACGGG CTCAAATACC

2651 TGTTCCCCGG GGAGTGCCAG

TACGTTCTGG TGCAGGATTA CTGCGGCAGT

2701 AACCCTGGGA CCTTTCGGAT

CCTAGTGGGG AATAAGGGAT GCAGCCACCC

2751 CTCAGTGAAA TGCAAGAAAC

GGGTCACCAT CCTGGTGGAG GGAGGAGAGA

2801 TTGAGCTGTT TGACGGGGAG

GTGAATGTGA AGAGGCCCAT GAAGGATGAG

2851 ACTCACTTTG AGGTGGTGGA

GTCTGGCCGG TACATCATTC TGCTGCTGGG

2901 CAAAGCCCTC TCCGTGGTCT

GGGACCGCCA CCTGAGCATC TCCGTGGTCC

2951 TGAAGCAGAC ATACCAGGAG

AAAGTGTGTG GCCTGTGTGG GAATTTTGAT

3001 GGCATCCAGA ACAATGACCT

CACCAGCAGC AACCTCCAAG TGGAGGAAGA

3051 CCCTGTGGAC TTTGGGAACT

CCTGGAAAGT GAGCTCGCAG TGTGCTGACA

3101 CCAGAAAAGT GCCTCTGGAC

TCATCCCCTG CCACCTGCCA TAACAACATC

3151 ATGAAGCAGA CGATGGTGGA

TTCCTCCTGT AGAATCCTTA CCAGTGACGT

3201 CTTCCAGGAC TGCAACAAGC

TGGTGGACCC CGAGCCATAT CTGGATGTCT

3251 GCATTTACGA CACCTGCTCC

TGTGAGTCCA TTGGGGACTG CGCCTGCTTC

3301 TGCGACACCA TTGCTGCCTA

TGCCCACGTG TGTGCCCAGC ATGGCAAGGT

3351 GGTGACCTGG AGGACGGCCA

CATTGTGCCC CCAGAGCTGC GAGGAGAGGA

3401 ATCTCCGGGA GAACGGGTAT

GAGTGTGAGT GGCGCTATAA CAGCTGTGCA

3451 CCTGCCTGTC AAGTCACGTG

TCAGCACCCT GAGCCACTGG CCTGCCCTGT

3501 GCAGTGTGTG GAGGGCTGCC ATGCCCACTG CCCTCCAGGG AAAATCCTGG

3551 ATGAGCTTTT GCAGACCTGC

GTTGACCCTG AAGACTGTCC AGTGTGTGAG

3601 GTGGCTGGCC GGCGTTTTGC

CTCAGGAAAG AAAGTCACCT TGAATCCCAG

3651 TGACCCTGAG CACTGCCAGA

TTTGCCACTG TGATGTTGTC AACCTCACCT

3701 GTGAAGCCTG CCAGGAGCCG

GGAGGCCTGG TGGTGCCTCC CACAGATGCC

3751 CCGGTGAGCC CCACCACTCT

GTATGTGGAG GACATCTCGG AACCGCCGTT

3801 GCACGATTTC TACTGCAGCA

GGCTACTGGA CCTGGTCTTC CTGCTGGATG

3851 GCTCCTCCAG GCTGTCCGAG

GCTGAGTTTG AAGTGCTGAA GGCCTTTGTG

3901 GTGGACATGA TGGAGCGGCT

GCGCATCTCC CAGAAGTGGG TCCGCGTGGC

3951 CGTGGTGGAG TACCACGACG

GCTCCCACGC CTACATCGGG CTCAAGGACC

4001 GGAAGCGACC GTCAGAGCTG

CGGCGCATTG CCAGCCAGGT GAAGTATGCG

4051 GGCAGCCAGG TGGCCTCCAC

CAGCGAGGTC TTGAAATACA CACTGTTCCA

4101 AATCTTCAGC AAGATCGACC

GCCCTGAAGC CTCCCGCATC GCCCTGCTCC

4151 TGATGGCCAG CCAGGAGCCC

CAACGGATGT CCCGGAACTT TGTCCGCTAC

4201 GTCCAGGGCC TGAAGAAGAA

GAAGGTCATT GTGATCCCGG TGGGCATTGG

4251 GCCCCATGCC AACCTCAAGC

AGATCCGCCT CATCGAGAAG CAGGCCCCTG

4301 AGAACAAGGC CTTCGTGCTG

AGCAGTGTGG ATGAGCTGGA GCAGCAAAGG

4351 GACGAGATCG TTAGCTACCT

CTGTGACCTT GCCCCTGAAG CCCCTCCTCC

4401 TACTCTGCCC CCCGACATGG

CACAAGTCAC TGTGGGCCCG GGGCTCTTGG

4451 GGGTTTCGAC CCTGGGGCCC

AAGAGGAACT CCATGGTTCT GGATGTGGCG

4501 TTCGTCCTGG AAGGATCGGA

CAAAATTGGT GAAGCCGACT TCAACAGGAG

4551 CAAGGAGTTC ATGGAGGAGG

TGATTCAGCG GATGGATGTG GGCCAGGACA

4601 GCATCCACGT CACGGTGCTG

CAGTACTCCT ACATGGTGAC CGTGGAGTAC

4651 CCCTTCAGCG AGGCACAGTC

CAAAGGGGAC ATCCTGCAGC GGGTGCGAGA

4701 GATCCGCTAC CAGGGCGGCA

ACAGGACCAA CACTGGGCTG GCCCTGCGGT

4751 ACCTCTCTGA CCACAGCTTC

TTGGTCAGCC AGGGTGACCG GGAGCAGGCG 4801 CCCAACCTGG TCTACATGGT

CACCGGAAAT CCTGCCTCTG ATGAGATCAA

4851 GAGGCTGCCT GGAGACATCC

AGGTGGTGCC CATTGGAGTG GGCCCTAATG

4901 CCAACGTGCA GGAGCTGGAG

AGGATTGGCT GGCCCAATGC CCCTATCCTC

4951 ATCCAGGACT TTGAGACGCT

CCCCCGAGAG GCTCCTGACC TGGTGCTGCA

5001 GAGGTGCTGC TCCGGAGAGG

GGCTGCAGAT CCCCACCCTC TCCCCTGCAC

5051 CTGACTGCAG CCAGCCCCTG

GACGTGATCC TTCTCCTGGA TGGCTCCTCC

5101 AGTTTCCCAG CTTCTTATTT

TGATGAAATG AAGAGTTTCG CCAAGGCTTT

5151 CATTTCAAAA GCCAATATAG

GGCCTCGTCT CACTCAGGTG TCAGTGCTGC

5201 AGTATGGAAG CATCACCACC

ATTGACGTGC CATGGAACGT GGTCCCGGAG

5251 AAAGCCCATT TGCTGAGCCT

TGTGGACGTC ATGCAGCGGG AGGGAGGCCC

5301 CAGCCAAATC GGGGATGCCT

TGGGCTTTGC TGTGCGATAC TTGACTTCAG

5351 AAATGCATGG TGCCAGGCCG

GGAGCCTCAA AGGCGGTGGT CATCCTGGTC

5401 ACGGACGTCT CTGTGGATTC

AGTGGATGCA GCAGCTGATG CCGCCAGGTC

5451 CAACAGAGTG ACAGTGTTCC

CTATTGGAAT TGGAGATCGC TACGATGCAG

5501 CCCAGCTACG GATCTTGGCA

GGCCCAGCAG GCGACTCCAA CGTGGTGAAG

5551 CTCCAGCGAA TCGAAGACCT

CCCTACCATG GTCACCTTGG GCAATTCCTT

5601 CCTCCACAAA CTGTGCTCTG

GATTTGTTAG GATTTGCATG GATGAGGATG

5651 GGAATGAGAA GAGGCCCGGG

GACGTCTGGA CCTTGCCAGA CCAGTGCCAC

5701 ACCGTGACTT GCCAGCCAGA

TGGCCAGACC TTGCTGAAGA GTCATCGGGT

5751 CAACTGTGAC CGGGGGCTGA

GGCCTTCGTG CCCTAACAGC CAGTCCCCTG

5801 TTAAAGTGGA AGAGACCTGT

GGCTGCCGCT GGACCTGCCC CTGYGTGTGC

5851 ACAGGCAGCT CCACTCGGCA

CATCGTGACC TTTGATGGGC AGAATTTCAA

5901 GCTGACTGGC AGCTGTTCTT

ATGTCCTATT TCAAAACAAG GAGCAGGACC

5951 TGGAGGTGAT TCTCCATAAT

GGTGCCTGCA GCCCTGGAGC AAGGCAGGGC

6001 TGCATGAAAT CCATCGAGGT

GAAGCACAGT GCCCTCTCCG TCGAGSTGCA

6051 CAGTGACATG GAGGTGACGG TGAATGGGAG ACTGGTCTCT GTTCCTTACG

6101 TGGGTGGGAA CATGGAAGTC

AACGTTTATG GTGCCATCAT GCATGAGGTC

6151 AGATTCAATC ACCTTGGTCA

CATCTTCACA TTCACTCCAC AAAACAATGA

6201 GTTCCAACTG CAGCTCAGCC

CCAAGACTTT TGCTTCAAAG ACGTATGGTC

6251 TGTGTGGGAT CTGTGATGAG

AACGGAGCCA ATGACTTCAT GCTGAGGGAT

6301 GGCACAGTCA CCACAGACTG

GAAAACACTT GTTCAGGAAT GGACTGTGCA

6351 GCGGCCAGGG CAGACGTGCC

AGCCCATCCT GGAGGAGCAG TGTCTTGTCC

6401 CCGACAGCTC CCACTGCCAG

GTCCTCCTCT TACCACTGTT TGCTGAATGC

6451 CACAAGGTCC TGGCTCCAGC

CACATTCTAT GCCATCTGCC AGCAGGACAG

6501 TTGCCACCAG GAGCAAGTGT

GTGAGGTGAT CGCCTCTTAT GCCCACCTCT

6551 GTCGGACCAA CGGGGTCTGC

GTTGACTGGA GGACACCTGA TTTCTGTGCT

6601 ATGTCATGCC CACCATCTCT

GGTCTACAAC CACTGTGAGC ATGGCTGTCC

6651 CCGGCACTGT GATGGCAACG

TGAGCTCCTG TGGGGACCAT CCCTCCGAAG

6701 GCTGTTTCTG CCCTCCAGAT

AAAGTCATGT TGGAAGGCAG CTGTGTCCCT

6751 GAAGAGGCCT GCACTCAGTG

CATTGGTGAG GATGGAGTCC AGCACCAGTT

6801 CCTGGAAGCC TGGGTCCCGG

ACCACCAGCC CTGTCAGATC TGCACATGCC

6851 TCAGCGGGCG GAAGGTCAAC

TGCACAACGC AGCCCTGCCC CACGGCCAAA

6901 GCTCCCACGT GTGGCCTGTG

TGAAGTAGCC CGCCTCCGCC AGAATGCAGA

6951 CCAGTGCTGC CCCGAGTATG

AGTGTGTGTG TGACCCAGTG AGCTGTGACC

7001 TGCCCCCAGT GCCTCACTGT

GAACGTGGCC TCCAGCCCAC ACTGACCAAC

7051 CCTGGCGAGT GCAGACCCAA

CTTCACCTGC GCCTGCAGGA AGGAGGAGTG

7101 CAAAAGAGTG TCCCCACCCT

CCTGCCCCCC GCACCGTTTG CCCACCCTTC

7151 GGAAGACCCA GTGCTGTGAT

GAGTATGAGT GTGCCTGCAA CTGTGTCAAC

7201 TCCACAGTGA GCTGTCCCCT

TGGGTACTTG GCCTCAACCG CCACCAATGA

7251 CTGTGGCTGT ACCACAACCA

CCTGCCTTCC CGACAAGGTG TGTGTCCACC

7301 GAAGCACCAT CTACCCTGTG

GGCCAGTTCT GGGAGGAGGG CTGCGATGTG 7351 TGCACCTGCA CCGACATGGA

GGATGCCGTG ATGGGCCTCC GCGTGGCCCA

7401 GTGCTCCCAG AAGCCCTGTG

AGGACAGCTG TCGGTCGGGC TTCACTTACG

7451 TTCTGCATGA AGGCGAGTGC

TGTGGAAGGT GCCTGCCATC TGCCTGTGAG

7501 GTGGTGACTG GCTCACCGCG

GGGGGACTCC CAGTCTTCCT GGAAGAGTGT

7551 CGGCTCCCAG TGGGCCTCCC

CGGAGAACCC CTGCCTCATC AATGAGTGTG

7601 TCCGAGTGAA GGAGGAGGTC

TTTATACAAC AAAGGAACGT CTCCTGCCCC

7651 CAGCTGGAGG TCCCTGTCTG

CCCCTCGGGC TTTCAGCTGA GCTGTAAGAC

7701 CTCAGCGTGC TGCCCAAGCT

GTCGCTGTGA GCGCATGGAG GCCTGCATGC

7751 TCAATGGCAC TGTCATTGGG

CCCGGGAAGA CTGTGATGAT CGATGTGTGC

7801 ACGACCTGCC GCTGCATGGT

GCAGGTGGGG GTCATCTCTG GATTCAAGCT

7851 GGAGTGCAGG AAGACCACCT

GCAACCCCTG CCCCCTGGGT TACAAGGAAG

7901 AAAATAACAC AGGTGAATGT

TGTGGGAGAT GTTTGCCTAC GGCTTGCACC

7951 ATTCAGCTAA GAGGAGGACA

GATCATGACA CTGAAGCGTG ATGAGACGCT

8001 CCAGGATGGC TGTGATACTC

ACTTCTGCAA GGTCAATGAG AGAGGAGAGT

8051 ACTTCTGGGA GAAGAGGGTC

ACAGGCTGCC CACCCTTTGA TGAACACAAG

8101 TGTCTTGCTG AGGGAGGTAA

AATTATGAAA ATTCCAGGCA CCTGCTGTGA

8151 CACATGTGAG GAGCCTGAGT

GCAACGACAT CACTGCCAGG CTGCAGTATG

8201 TCAAGGTGGG AAGCTGTAAG

TCTGAAGTAG AGGTGGATAT CCACTACTGC

8251 CAGGGCAAAT GTGCCAGCAA

AGCCATGTAC TCCATTGACA TCAACGATGT

8301 GCAGGACCAG TGCTCCTGCT

GCTCTCCGAC ACGGACGGAG CCCATGCAGG

8351 TGGCCCTGCA CTGCACCAAT

GGCTCTGTTG TGTACCATGA GGTTCTCAAT

8401 GCCATGGAGT GCAAATGCTC

CCCCAGGAAG TGCAGCAAGT GA

The VWF fragment as used herein can be a VWF fragment comprising aain and a D3 domain of VWF, wherein the VWF fragment binds to Factor VIII (FVIII) and inhibits binding of endogenous VWF (full-length VWF) to FVIII. The VWF fragment comprising the D' domain and the D3 domain can further comprise a VWF domain selected from the group consisting of an A 1 domain, an A2 domain, an A3 domain, a Dl domain, a D2 domain, a D4 domain, a Bl domain, a B2 domain, a B3 domain, a CI domain, a C2 domain, a CK domain, one or more fragments thereof, and any combinations thereof. In one

embodiment, a VWF fragment comprises, consists essentially of, or consists of: (1) the D' and D3 domains of VWF or fragments thereof; (2) the Dl, D', and D3 domains of VWF or fragments thereof; (3) the D2, D', and D3 domains of VWF or fragments thereof; (4) the Dl, D2, D', and D3 domains of VWF or fragments thereof; or (5) the Dl, D2, D', D3, and Al domains of VWF or fragments thereof. The VWF fragment described herein does not contain a site binding to a VWF clearance receptor. In another embodiment, the VWF fragment described herein is not amino acids 764 to 1274 of SEQ ID NO: 2. The VWF fragment of the present invention can comprise any other sequences linked to or fused to the VWF fragment. For example, a VWF fragment described herein can further comprise a signal peptide.

In one embodiment, the VWF fragment binds to or is associated with a

FVIII protein. By binding to or associating with a FVIII protein, a VWF fragment of the invention protects FVIII from protease cleavage and FVIII activation, stabilizes the heavy chain and light chain of FVIII, and prevents clearance of FVIII by scavenger receptors. In another embodiment, the VWF fragment binds to or associates with a FVIII protein and blocks or prevents binding of the FVIII protein to phospholipid and activated Protein C. By preventing or inhibiting binding of the FVIII protein with endogenous, full-length VWF, the VWF fragment of the invention reduces the clearance of FVIII by VWF clearance receptors and thus extends half-life of the FVIII protein. In one embodiment, the half-life extension of a FVIII protein is thus due to the binding of or associating with the VWF fragment lacking a VWF clearance receptor binding site to the FVIII protein and shielding or protecting of the FVIII protein by the VWF fragment from endogenous VWF which contains the VWF clearance receptor binding site. The FVIII protein bound to or protected by the VWF fragment can also allow recycling of a FVIII protein. By eliminating the VWF clearance pathway receptor binding sites contained in the full length VWF molecule, the FVIII/VWF heterodimers of the invention are shielded from the VWF clearance pathway, further extending FVIII half-life.

[0144] In one embodiment, a VWF fragment of the present invention comprises the D' domain and the D3 domain of VWF, wherein the D' domain is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764 to 866 of SEQ ID NO: 2, wherein the VWF fragment prevents binding of endogenous VWF to FVIII. In another embodiment, a VWF fragment comprises the D' domain and the D3 domain of VWF, wherein the D3 domain is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 867 to 1240 of SEQ ID NO: 2, wherein the VWF fragment prevents binding of endogenous VWF to FVIII. In some embodiments, a VWF fragment described herein comprises, consists essentially of, or consists of the D' domain and D3 domain of VWF, which are at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764 to 1240 of SEQ ID NO: 2, wherein the VWF fragment prevents binding of endogenous VWF to FVIII. In other embodiments, a VWF fragment comprises, consists essentially of, or consists of the D l , D2, D*, and D3 domains at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 23 to 1240 of SEQ ID NO: 2, wherein the VWF fragment prevents binding of endogenous VWF to FVIII. In still other embodiments, the VWF fragment further comprises a signal peptide operably linked thereto.

[0145] In some embodiments, a VWF fragment of the invention consists

essentially of or consists of (1) the D'D3 domain, the D1D'D3 domain, D2D'D3 domain, or D1D2DO3 domain and (2) an additional VWF sequence up to about 10 amino acids (e.g., any sequences from amino acids 764 to 1240 of SEQ ID NO: 2 to amino acids 764 to 1250 of SEQ ID NO: 2), up to about 15 amino acids (e.g., any sequences from amino acids 764 to 1240 of SEQ ID NO: 2 to amino acids 764 to 1255 of SEQ ID NO: 2), up to about 20 amino acids (e.g., any sequences from amino acids 764 to 1240 of SEQ ID NO: 2 to amino acids 764 to 1260 of SEQ ID NO: 2), up to about 25 amino acids (e.g., any sequences from amino acids 764 to 1240 of SEQ ID NO: 2 to amino acids 764 to 1265 of SEQ ID NO: 2), or up to about 30 amino acids (e.g., any sequences from amino acids 764 to 1240 of SEQ ID NO: 2 to amino acids 764 to 1260 of SEQ ID NO: 2). In a particular embodiment, the VWF fragment comprising or consisting essentially of the D' domain and the D3 domain is neither amino acids 764 to 1274 of SEQ ID NO: 2 nor the full-length mature VWF. In some embodiments, the D1D2 domain is expressed in trans with the D'D3 domain. In some embodiments, the D1D2 domain is expressed in cis with the D'D3 domain.

[0146] In other embodiments, the VWF fragment comprising the D'D3 domains linked to the D1D2 domains further comprises an intracellular cleavage site, e.g., (a cleavage site by PACE (furin) or PC5), allowing cleavage of the D1D2 domains from the D'D3 domains upon expression. Non-limiting examples of the intracellular cleavage site are disclosed elsewhere herein.

[0147] In yet other embodiments, a VWF fragment comprises the D' domain and the D3 domain, but does not comprise an amino acid sequence selected from the group consisting of (1) amino acids 1241 to 2813 of SEQ ID NO: 2, (2) amino acids 1270 to amino acids 2813 of SEQ ID NO: 2, (3) amino acids 1271 to amino acids 2813 of SEQ ID NO: 2, (4) amino acids 1272 to amino acids 2813 of SEQ ID NO: 2, (5) amino acids 1273 to amino acids 2813 of SEQ ID NO: 2, (6) amino acids 1274 to amino acids 2813 of SEQ ID NO: 2, and any combinations thereof.

[0148] In still other embodiments, a VWF fragment of the present invention

comprises, consists essentially of, or consists of an amino acid sequence corresponding to the D' domain, D3 domain, and Al domain, wherein the amino acid sequence is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acid 764 to 1479 of SEQ ID NO: 2, wherein the VWF fragment prevents binding of endogenous VWF to FVIII. In a particular embodiment, the VWF fragment is not amino acids 764 to 1274 of SEQ ID NO: 2.

[0149] In some embodiments, a VWF fragment of the invention comprises the D' domain and the D3 domain, but does not comprise at least one VWF domain selected from the group consisting of (1) an Al domain, (2) an A2 domain, (3) an A3 domain, (4) a D4 domain, (5) a Bl domain, (6) a B2 domain, (7) a B3 domain, (8) a CI domain, (9) a C2 domain, (10) a CK domain, (11) a CK domain and C2 domain, (12) a CK domain, a C2 domain, and a CI domain, (13) a CK domain, a C2 domain, a CI domain, a B3 domain, (14) a CK domain, a C2 domain, a CI domain, a B3 domain, a B2 domain, (15) a CK domain, a C2 domain, a CI domain, a B3 domain, a B2 domain, and a Bl domain, (16) a CK domain, a C2 domain, a CI domain, a B3 domain, a B2 domain, a Bl domain, and a D4 domain, (17) a CK domain, a C2 domain, a CI domain, a B3 domain, a B2 domain, a Bl domain, a D4 domain, and an A3 domain, (18) a CK domain, a C2 domain, a CI domain, a B3 domain, a B2 domain, a Bl domain, a D4 domain, an A3 domain, and an A2 domain, (19) a CK domain, a C2 domain, a CI domain, a B3 domain, a B2 domain, a Bl domain, a D4 domain, an A3 domain, an A2 domain, and an Al domain, and (20) any combinations thereof.

[0150] In yet other embodiments, the VWF fragment comprises the D'D3 domains and one or more domains or modules. Examples of such domains or modules include, but are not limited to, the domains and modules disclosed in Zhour et al, Blood published online April 6, 2012: DOI 10.1182/blood-2012-01-405134. For example, the VWF fragment can comprise the D'D3 domain and one or more domains or modules selected from the group consisting of Al domain, A2 domain, A3 domain, D4N module, VWD4 module, C8-4 module, TIL-4 module, CI module, C2 module, C3 module, C4 module, C5 module, C5 module, C6 module, and any combinations thereof.

[0151] In still other embodiments, the VWF fragment is linked to a heterologous moiety, wherein the heterologous moiety is linked to the N-terminus or the C- terminus of the VWF fragment or inserted immediately downstream of one or more amino acids (e.g., one or more XTEN insertion sites) in the FVIII protein in the VWF fragment. For example, the insertion sites for the heterologous moiety in the VWF fragment can be in the D' domain, the D3 domain, or both. The heterologous moiety can be a half-life extender.

[0152] In certain embodiments, a VWF fragment of the invention forms a

multimer, e.g., dimer, trimer, tetramer, pentamer, hexamer, heptamer, or the higher order multimers. In other embodiments, the VWF fragment is a monomer having only one VWF fragment. In some embodiments, the VWF fragment of the present invention can have one or more amino acid substitutions, deletions, additions, or modifications. In one embodiment, the VWF fragment can include amino acid substitutions, deletions, additions, or modifications such that the VWF fragment is not capable of forming a disulfide bond or forming a dimer or a multimer. In another embodiment, the amino acid substitution is within the D' domain and the D3 domain. In a particular embodiment, a VWF fragment of the invention contains at least one amino acid substitution at a residue corresponding to residue 1099, residue 1142, or both residues 1099 and 1142 of SEQ ID NO: 2. The at least one amino acid substitution can be any amino acids that are not occurring naturally in the wild type VWF. For example, the amino acid substitution can be any amino acids other than cysteine, e.g., Isoleucine, Alanine, Leucine, Asparagine, Lysine, Aspartic acid, Methionine, Phenylalanine, Glutamic acid, Threonine, Glutamine, Tryptophan, Glycine, Valine, Proline, Serine, Tyrosine, Arginine, or Histidine. In another example, the amino acid substitution has one or more amino acids that prevent or inhibit the VWF fragments from forming multimers.

[0153] In certain embodiments, the VWF fragment useful herein can be further modified to improve its interaction with FVIII, e.g., to improve binding affinity to FVIII. As a non-limiting example, the VWF fragment comprises a serine residue at the residue corresponding to amino acid 764 of SEQ ID NO: 2 and a lysine residue at the residue corresponding to amino acid 773 of SEQ ID NO: 2.

Residues 764 and/or 773 can contribute to the binding affinity of the VWF fragments to FVIII. In other embodiments, the VWF fragments useful for the invention can have other modifications, e.g., the protein can be pegylated, glycosylated, hesylated, or polysialylated.

B) XTEN Sequences

[0154] As used here "XTEN sequence" refers to extended length polypeptides with non-naturally occurring, substantially non-repetitive sequences that are composed mainly of small hydrophilic amino acids, with the sequence having a low degree or no secondary or tertiary structure under physiologic conditions. As a chimeric protein partner, XTENs can serve as a carrier, conferring certain desirable pharmacokinetic, physicochemical and pharmaceutical properties when linked to a VWF fragment or a FVIII sequence of the invention to create a chimeric protein. Such desirable properties include but are not limited to enhanced pharmacokinetic parameters and solubility characteristics. As used herein, "XTEN" specifically excludes antibodies or antibody fragments such as single-chain antibodies or Fc fragments of a light chain or a heavy chain. [0155] In some embodiments, the XTEN sequence of the invention is a peptide or a polypeptide having greater than about 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, or 2000 amino acid residues. In certain

embodiments, XTEN is a peptide or a polypeptide having greater than about 20 to about 3000 amino acid residues, greater than 30 to about 2500 residues, greater than 40 to about 2000 residues, greater than 50 to about 1500 residues, greater than 60 to about 1000 residues, greater than 70 to about 900 residues, greater than 80 to about 800 residues, greater than 90 to about 700 residues, greater than 100 to about 600 residues, greater than 110 to about 500 residues, or greater than 120 to about 400 residues.

[0156] The XTEN sequence of the invention can comprise one or more sequence motif of 9 to 14 amino acid residues or an amino acid sequence at least 80%, 90%>, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence motif, wherein the motif comprises, consists essentially of, or consists of 4 to 6 types of amino acids selected from the group consisting of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P). See US 2010- 0239554 Al .

[0157] In some embodiments, the XTEN comprises non-overlapping sequence motifs in which about 80%, or at least about 85%, or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%), or about 98%>, or about 99% or about 100% of the sequence consists of multiple units of non-overlapping sequences selected from a single motif family selected from Table 2A, resulting in a family sequence. As used herein, "family" means that the XTEN has motifs selected only from a single motif category from Table 2A; i.e., AD, AE, AF, AG, AM, AQ, BC, or BD XTEN, and that any other amino acids in the XTEN not from a family motif are selected to achieve a needed property, such as to permit incorporation of a restriction site by the encoding nucleotides, incorporation of a cleavage sequence, or to achieve a better linkage to FVIII or VWF. In some embodiments of XTEN families, an XTEN sequence comprises multiple units of non-overlapping sequence motifs of the AD motif family, or of the AE motif family, or of the AF motif family, or of the AG motif family, or of the AM motif family, or of the AQ motif family, or of the BC family, or of the BD family, with the resulting XTEN exhibiting the range of homology described above. In other embodiments, the XTEN comprises multiple units of motif sequences from two or more of the motif families of Table 2A. These sequences can be selected to achieve desired physical/chemical characteristics, including such properties as net charge, hydrophilicity, lack of secondary structure, or lack of repetitiveness that are conferred by the amino acid composition of the motifs, described more fully below. In the embodiments hereinabove described in this paragraph, the motifs incorporated into the XTEN can be selected and assembled using the methods described herein to achieve an XTEN of about 36 to about 3000 amino acid residues.

Table 2A. XTEN Sequence Motifs of 12 Amino Acids and Motif Families

Motif I^'aniih · MOTH SKQl KM l.

AD GESPGGSSGSES

AD GSEGSSGPGESS

AD GSSESGSSEGGP

AD GSGGEPSESGSS

AE, AM GSPAGSPTSTEE

AE, AM, AQ GSEPATSGSETP

AE, AM, AQ GTSESATPESGP

AE, AM, AQ GTSTEPSEGSAP

AF, AM GSTSESPSGTAP

AF, AM GTSTPESGSASP

AF, AM GTSPSGESSTAP

AF, AM GSTSSTAESPGP

AG, AM GTPGSGTASSSP

AG, AM GSSTPSGATGSP

AG, AM GSSPSASTGTGP

AG, AM GASPGTSSTGSP

AQ GEPAGSPTSTSE

AQ GTGEPSSTPASE

AQ GSGPSTESAPTE

AQ GSETPSGPSETA

AQ GPSETSTSEPGA

AQ GSPSEPTEGTSA

BC GSGASEPTSTEP

BC GSEPATSGTEPS

BC GTSEPSTSEPGA

BC GTSTEPSEPGSA

BD GSTAGSETSTEA

BD GSETATSGSETA

BD GTSESATSESGA

BD GTSTEASEGSAS • Denotes individual motif sequences that, when used together in various permutations, results in a "family sequence"

[0158] XTEN can have varying lengths for insertion into or linkage to FVIII or

VWF. In one embodiment, the length of the XTEN sequence(s) is chosen based on the property or function to be achieved in the fusion protein. Depending on the intended property or function, XTEN can be short or intermediate length sequence or longer sequence that can serve as carriers. In certain embodiments, the XTEN include short segments of about 6 to about 99 amino acid residues, intermediate lengths of about 100 to about 399 amino acid residues, and longer lengths of about 400 to about 1000 and up to about 3000 amino acid residues. Thus, the XTEN inserted into or linked to FVIII or VWF can have lengths of about 6, about 12, about 36, about 40, about 42, about 72, about 96, about 144, about 288, about 400, about 500, about 576, about 600, about 700, about 800, about 864, about 900, about 1000, about 1500, about 2000, about 2500, or up to about 3000 amino acid residues in length. In other embodiments, the XTEN sequences is about 6 to about 50, about 50 to about 100, about 100 to 150, about 150 to 250, about 250 to 400, about 400 to about 500, about 500 to about 900, about 900 to 1500, about 1500 to 2000, or about 2000 to about 3000 amino acid residues in length. The precise length of an XTEN inserted into or linked to FVIII or VWF can vary without adversely affecting the activity of the FVIII or VWF. In one embodiment, one or more of the XTEN used herein has 36 amino acids, 42 amino acids, 72 amino acids, 144 amino acids, 288 amino acids, 576 amino acids, or 864 amino acids in length and can be selected from one or more of the XTEN family sequences; i.e., AD, AE, AF, AG, AM, AQ, BC or BD.

[0159] In some embodiments, the XTEN sequence used in the invention is at least

60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a sequence selected from the group consisting of AE42, AG42, AE48, AM48, AE72, AG72, AE108, AG108, AE144, AF144, AG144, AE180, AG180, AE216, AG216, AE252, AG252, AE288, AG288, AE324, AG324, AE360, AG360, AE396, AG396, AE432, AG432, AE468, AG468, AE504, AG504, AF504, AE540, AG540, AF540, AD576, AE576, AF576, AG576, AE612, AG612, AE624, AE648, AG648, AG684, AE720, AG720, AE756, AG756, AE792, AG792, AE828, AG828, AD836, AE864, AF864, AG864, AM875, AE912, AM923, AM1318, BC864, BD864, AE948, AE1044, AE1 140, AE1236, AE1332, AE1428, AE1524, AE1620, AE1716, AE1812, AE1908, AE2004A, AG948, AG1044, AG1 140, AG1236, AG1332, AG1428, AG1524, AG1620, AG1716, AG1812, AG1908, and AG2004. See US 2010- 0239554 Al .

[0160] In one embodiment, the XTEN sequence is at least 60%, 70%, 80%, 90%,

95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of AE42 (SEQ ID NO: 36), AE72 (SEQ ID NO: 127), AE144 2A (SEQ IDNO: 128), AE144 3B (SEQ ID NO: 129), AE144 4A (SEQ ID NO: 130), AE144 5A (SEQ IDNO: 131), AE144 6B (SEQ IDNO: 132), AG144 A (SEQ ID NO: 133), AG144 B (SEQ IDNO: 134), AG144 C (SEQ ID NO: 135), AG144 F (SEQ IDNO: 136), AE864 (SEQ ID NO: 43), AE576 (SEQ ID NO: 41), AE288 (SEQ IDNO: 39), AE288 2 (SEQ ID NO: 137), AE144 (SEQ ID NO: 37), AG864 (SEQ ID NO: 44), AG576 (SEQ ID NO: 42), AG288 (SEQ ID NO: 40), AG 144 (SEQ ID NO: 38), and any combinations thereof.

[0161] In some embodiments, less than 100% of amino acids of an XTEN are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), or less than 100% of the sequence consists of the sequence motifs from Table 2 A or the XTEN sequences of Table 2B. In such embodiments, the remaining amino acid residues of the XTEN are selected from any of the other 14 natural L-amino acids, but may be preferentially selected from hydrophilic amino acids such that the XTEN sequence contains at least about 90%>, 91 >, 92%>, 93%>, 94%, 95%, 96%, 97%, 98%, or at least about 99% hydrophilic amino acids. The content of hydrophobic amino acids in the XTEN utilized in the conjugation constructs may be less than 5%>, or less than 2%>, or less than 1%> hydrophobic amino acid content. Hydrophobic residues that are less favored in construction of XTEN include tryptophan, phenylalanine, tyrosine, leucine, isoleucine, valine, and methionine. Additionally, XTEN sequences may contain less than 5% or less than 4% or less than 3% or less than 2% or less than 1 % or none of the following amino acids: methionine (for example, to avoid oxidation), or asparagine and glutamine (to avoid desamidation).

[0162] In another embodiment, the XTEN sequence is selected from the group consisting of AE42 (SEQ ID NO: 36), AE72 (SEQ ID NO: 127), AE144 2A (SEQ IDNO: 128), AE144 3B (SEQ ID NO: 129), AE144 4A (SEQ ID NO: 130), AE144 5A (SEQ IDNO: 131), AE144 6B (SEQ IDNO: 132), AG144 A (SEQ ID NO: 133), AG144 B (SEQ IDNO: 134), AG144 C (SEQ ID NO: 135), AG144 F (SEQ IDNO: 136), AE864 (SEQ ID NO: 43), AE576 (SEQ ID NO: 41), AE288 (SEQ IDNO: 39), AE288 2 (SEQ ID NO: 137), AE144 (SEQ ID NO: 37), AG864 (SEQ ID NO: 44), AG576 (SEQ ID NO: 42), AG288 (SEQ ID NO: 40), AG 144 (SEQ ID NO: 38), and any combinations thereof. In a specific

embodiment, the XTEN sequence is AE288. The amino acid sequences for certain XTEN sequences of the invention are shown in Table 2B.

TABLE 2B. XTEN Sequences

XTEN Amino Acid Sequence

AE42 GAPGSPAGSP S EEG SESA PESGPGSEPA SGSE PASS

SEQ ID NO:

36

AE72 GAPTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPA

SEQ ID NO: TSGSETPGTSESATPESGPGTSTEPSEGSAPGASS

127

AE144 GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTE

SEQ ID EGTSTEPSEG

NO:37 SAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSE

GSAPGTSESA

PESGPGSEPATSGSETPGTSTEPSEGSAP

AE144_2A TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAP

GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSET

(SEQ ID NO:

PGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPES

128) GPG

AE144 3B SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGP

GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA

(SEQ ID NO:

PGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS

129) APG

AE144_4A TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP

GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTE

(SEQ ID NO:

EGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGS

130) APG

AE144_5A TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP

GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESG

(SEQ ID NO:

PGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTST 131) EEG

AE144 6B TSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGP

GSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSA

(SEQ ID NO:

PGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGS

132) APG

AG 144 G PGSGTASSSPGSS PSGATGSPGSS PSAS GTGPGSS PSAS GTG

SEQ ID PGASPGTSST

NO:38 GSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSS

TGSPGSSPSA

STGTGPGTPGSGTASSSPGSSTPSGATGSP

AG144_A GAS PG SS GSPGSS PSAS GTGPGSS PSAS GTGPG PGSGTASSS

PGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASS

(SEQ ID NO:

SPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGS PGASPGTSST

133) GSP

AG144 B GTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSS

PGSSTPSGATGSPGSS PSAS TGTGPGSS PSAS TGTGPGSSTPSGATG

(SEQ ID NO:

SPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGS PGASPGTSST

134) GSP

AG144_C GTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGS

PGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTG

(SEQ ID NO:

SPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGS PGASPGTSST

135) GSP

AG144 F GSS PSAS TGTGPGSS PSAS TGTGPGASPGTSSTGSPGASPGTSSTGS

PGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGT

(SEQ ID NO:

GPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGS PGASPGTSST

136) GSP

AE288 GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSET

PGTSESATPESG

SEQ ID

PGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE NO:39 TPGTSESATPES

GPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPE SGPGTSESATPE

SGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT STEEGTSTEPSE

GSAPGT S TE P SEGSAPGSE PAT S GSETPGTSE SAT PESGPGT STEPS EGSAP

AE288_2 GS PAGS PT S TEEGT SE SAT PESGPGSE PATS GSETPGTSE SAT PESG

PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS

(SEQ ID NO:

APGT STEPSEGSAPGTSTEPSEGSAPGS PAGS PTS TEEGT STEPSEG

137) SAPGT SE SAT PE SGPGSE PATS GSETPGTSE SAT PESGPGSE PAT SG

SE T PGT SE SAT PE SGPGTSTEPSEGSAPGTSE SAT PESGPGS PAGS P T S TEEGS PAGS PT S TEEGS PAGS PTS TEEGT SE SAT PESGPGT STEP SEGSAP

AG288 PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG SEQ ID SPGTPGSGTASS

SPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGAT NO:40

GSPGSSPSASTG

TGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGA TGSPGSSPSAST

GTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSG ATGSPGSSPSAS

TGTGPGAS PG S S GSPGSS PSAS GTGPG PGSGTASSSPGS S PS GATGS

AE576 GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTE

EGTSTEPSEGSA

SEQ ID

PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSE NO:41 TPGSPAGSPTST

EEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTS TEEGTSTEPSEG

SAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATP ESGPGSEPATSG

SETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESAT PESGPGSPAGSP

T S TEEGT SE SAT PE S GPGSE PAT SGSET PGT SE SAT PESGPGT STEP SEGSAPGTSTEP

SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTE PSEGSAPGSPAG

S PT S TEEGT STEPSEGSAPGTSE SAT PES GPGSE PAT SGSET PGT SE SATPESGPGSEP

AT S GSE T PGT SE SAT PESGPGT STEPSEGSAPGTSE SAT PESGPGSP AGSPTSTEEGSP

AGS PT S TEEGSPAGSPTS TEEGT SE SAT PESGPGTSTEPSEGSAP

AG576 PGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGT

GPGSSTPSGATG

SEQ ID

SPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSST

NO:42 GSPGTPGSGTAS

SSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSAST GTGPGTPGSGTA

SSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSG ATGSPGSSTPSG

ATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPS GATGSPGSSTPS

GATGS PGSS PSAS TGTGPGAS PGT SSTGSPGAS PGT SSTGS PGT PGS GTASSSPGASPG

TSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPG SGTASSSPGSST

PSGATGS PGT PGSGTASSS PGS STPSGATGS PGT PGSGTASSS PGSS TPSGATGSPGSS

T PSGATGS PGSS PSAS TGTGPGSS PSAS TGTGPGAS PGT SSTGS PGT PGSGTASSSPGS

STPSGATGS PGS SPSASTGTGPGSSPSASTGTGPGASPGTSSTGS

AE864 GS PAGSPTS TEEGT SE SAT PESGPGTSTEPSEGSAPGSPAGSPTSTE

EGTSTEPSEGSA SEQ ID PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSE TPGSPAGSPTST NO:43

EEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTS TEEGTSTEPSEG

SAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATP ESGPGSEPATSG

SETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESAT PESGPGSPAGSP

T S TEEGT SE SAT PE S GPGSE PAT SGSETPGTSE SAT PESGPGT STEP SEGSAPGTSTEP

SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTE PSEGSAPGSPAG

S PT S TEEGT STEPSEGSAPGTSE SAT PES GPGSE PAT SGSETPGTSE SATPESGPGSEP

AT S GSE T PGTSE SAT PESGPGT STEPSEGSAPGTSE SAT PESGPGSP AGSPTSTEEGSP

AGS PT STEEGSPAGSPTS TEEGT SE SAT PESGPGTSTEPSEGSAPGT SESATPESGPGS

E PAT S GSE T PGTSE SAT PES GPGSE PAT SGSETPGTSE SAT PESGPG TSTEPSEGSAPG

S PAGS PT S TEEGT SE SAT PES GPGSE PAT SGSETPGTSE SAT PESGP GSPAGSPTSTEE

GS PAGS PT S TEEGT STEPSEGSAPGTSE SAT PESGPGTSE SAT PESG PGTSESATPESG

PGSE PAT S GSE TPGSE PATS GSE TPGS PAGS PTS TEEGT STEPSEGS APGTSTEPSEGS

APGSE PAT S GSE T PGTSE SAT PESGPGT STEPSEGS AP

AG864 GAS PGTSSTGSPGSS PSAS TGTGPGSS PSAS TGTGPGTPGSGTASSS

PGSSTPSGATGS

SEQ ID

PGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG

NO:44 SPGTPGSGTASS

SPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT GSPGASPGTSST

GSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAST GTGPGSSTPSGA

TGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS STGSPGTPGSGT

AS SSPGASPGTSSTGSPGASPGTSSTGSPGAS PGTSSTGSPGSS PSA STGTGPGTPGSG

TASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTP SGATGSPGSSTP

SGATGSPGASPGTSSTGSPGT PGSGTASSSPGS ST PSGATGSPGSST PSGATGSPGSST

PSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTP GSGTASSSPGAS

PGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGT PGSGTASSSPGS

STPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPG SSTPSGATGSPG

SSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSP GTPGSGTASSSP GSS PSGATGSPGSS PSAS GTGPGSS PSAS GTGPGASPG SS GS

PGASPGTSSTGS

PGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGT

GPGTPGSGTASS

SPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP

[0163] In further embodiments, the XTEN sequence used in the invention affects the physical or chemical property, e.g., pharmacokinetics, of the chimeric protein of the present invention. The XTEN sequence used in the present invention can exhibit one or more of the following advantageous properties: conformational flexibility, enhanced aqueous solubility, high degree of protease resistance, low immunogenicity, low binding to mammalian receptors, or increased hydrodynamic (or Stokes) radii. In a specific embodiment, the XTEN sequence linked to a FVIII protein in this invention increases pharmacokinetic properties such as longer terminal half-life or increased area under the curve (AUC), so that the chimeric protein described herein stays in vivo for an increased period of time compared to wild type FVIII. In further embodiments, the XTEN sequence used in this invention increases pharmacokinetic properties such as longer terminal half-life or increased area under the curve (AUC), so that FVIII protein stays in vivo for an increased period of time compared to wild type FVIII.

[0164] A variety of methods and assays can be employed to determine the

physical/chemical properties of proteins comprising the XTEN sequence. Such methods include, but are not limited to analytical centrifugation, EPR, HPLC-ion exchange, HPLC-size exclusion, HP LC -reverse phase, light scattering, capillary electrophoresis, circular dichroism, differential scanning calorimetry,

fluorescence, HPLC-ion exchange, HPLC-size exclusion, IR, NMR, Raman spectroscopy, refractometry, and UV/Visible spectroscopy. Additional methods are disclosed in Amau et al, Prot Expr and Purif 48, 1-13 (2006).

[0165] Additional examples of XTEN sequences that can be used according to the present invention and are disclosed in US Patent Publication Nos.2010/0239554 Al, 2010/0323956 Al, 2011/0046060 Al, 2011/0046061 Al, 2011/0077199 Al, or 2011/0172146 A 1, or International Patent Publication Nos. WO 2010091122 Al, WO 2010144502 A2, WO 2010144508 Al, WO 2011028228 Al, WO 2011028229 Al, or WO 2011028344 A2.

C) Factor VIII (FVIII) Protein [0166] "A FVIII protein" as used herein means a functional FVIII polypeptide in its normal role in coagulation, unless otherwise specified. The term a FVIII protein includes a functional fragment, variant, analog, or derivative thereof that retains the function of full-length wild-type Factor VIII in the coagulation pathway. "A FVIII protein" is used interchangeably with FVIII polypeptide (or protein) or FVIII. Examples of the FVIII functions include, but not limited to, an ability to activate coagulation, an ability to act as a cofactor for factor IX, or an

2__|_

ability to form a tenase complex with factor ΓΧ in the presence of Ca and phospholipids, which then converts Factor X to the activated form Xa. The FVIII protein can be the human, porcine, canine, rat, or murine FVIII protein. In addition, comparisons between FVIII from humans and other species have identified conserved residues that are likely to be required for function (Cameron et al, Thromb. Haemost. 79:317-22 (1998); US 6,251,632).

[0167] A number of tests are available to assess the function of the coagulation system: activated partial thromboplastin time (aPTT) test, chromogenic assay, ROTEM assay, prothrombin time (PT) test (also used to determine INR), fibrinogen testing (often by the Clauss method), platelet count, platelet function testing (often by PFA-100), TCT, bleeding time, mixing test (whether an abnormality corrects if the patient's plasma is mixed with normal plasma), coagulation factor assays, antiphospholipid antibodies, D-dimer, genetic tests (e.g., factor V Leiden, prothrombin mutation G20210A), dilute Russell's viper venom time (dRVVT), miscellaneous platelet function tests, thromboelastography (TEG or Sonoclot), thromboelastometry (TEM^®, e.g., ROTEM^®), or euglobulin lysis time (ELT).

[0168] The aPTT test is a performance indicator measuring the efficacy of both the "intrinsic" (also referred to the contact activation pathway) and the common coagulation pathways. This test is commonly used to measure clotting activity of commercially available recombinant clotting factors, e.g., FVIII or FIX. It is used in conjunction with prothrombin time (PT), which measures the extrinsic pathway.

[0169] ROTEM analysis provides information on the whole kinetics of

haemostasis: clotting time, clot formation, clot stability and lysis. The different parameters in thromboelastometry are dependent on the activity of the plasmatic coagulation system, platelet function, fibrinolysis, or many factors which influence these interactions. This assay can provide a complete view of secondary haemostasis.

The FVIII polypeptide and polynucleotide sequences are known, as are many functional fragments, mutants and modified versions. Examples of human FVIII sequences (full-length) are shown below.

TABLE 3. Amino Acid Sequence of Full-length Factor VIII

(Full-length FVIII (FVIII signal peptide underlined; FVIII heavy chain is double underlined; B domain is italicized; and FVIII light chain is in plain text)

Signal Peptide: (SEQ ID NO: 3)

Mature Factor VIII (SEQ ID NO: 4)*

SFSQMSREPS R QKQFN A T'IFENDI E.KTD PWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAI ESNNSlYYEMlYLiYRPQLEHSGEtiVbYYYiJSGL

TSNNLISTIPSDNLAAGTDNTSSLGPPSMPVEYDSGLDPTLFXFKKSSPLTESGGP LSLSEENNDSKLLESGLYINSQESSWGKNVSSTESGELFKGERAEGPALLTKENAL FKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPL IHDRMLMDKNA TA .,RLNHMSNKTTSSKNME^ QQKKEGPIPPDAQNPDMSFFKPL FLPESAR WIQR HGKNSLNSGGGPS PKQLVSLGPEKSVEGQNFLSEKNKVVVGKG EEYKDVGLKEMVFPSSRNLFL TNLDNLHENNTENQEKK1OEE1EKKETLIQENVV LPQ I HTVTG TKNFMKNIJ IELLS RONVEGSYDGAYAPVLQDFRSLNDSTNRTKKET

AEFSKKGEEENLEGLGNG KQIVEKYACTTE^LSPNTSGQ^R^Y RGERALEQ RL PLEETELEKEYI VDD TS QWSKNMKELTPS LTQIDYNEKEKGAITQSPLS D CLT RSHSYPQANRSPLP AKVSSEPSLRPYYL YRVLFQDNSSHLPAA.S YRKKESGVOE SSHELQGAKKNNLSLA IL TLEMTGDQREVGSLGYSA TNS VTYKKVENTVLPKPDL PKTSGKVELLPKVEYYQKDLFPTETSNGSPGHLDLVEGSLLGGTEGAJKWNEANR PGKVPF1E AYESSAXYY^>SKLIA:GYJAIYDN]LY PYIRAEVEDNIMVTFRNQASRPYSFYSSLI SYEEDQRQGAEPRKNFVKPNETKTY FWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQ VTVQEFALFF IFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYI MDTLPGLVMAQDQRIRWYLLSMGSNE IHS IHFSGHVFTVRKKEEYKMALYNLYP GVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDF QI ASGQYGQWAPKLARLHYSGS INAWS KEPFSWIKVDLLAPMI IHGIKTQGAR QKFSSLYI SQFI IMYSLDGKKWQTYRGNS GTLMVFFGNVDSSGIKHNIFNPPI I ARYIRLHPTHYS IRS LRMELMGCDLNSCSMPLGMESKAI SDAQI ASSYFTNMF ATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSM YVKEFLI SSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHP QSWVHQIALRMEVLGCEAQDLY

TABLE 4. Nucleotide Sequence Encoding Full-Length FVIII (SEQ ID NO

661 ATG

CAAATAGAGC TCTCCACCTG

721 CTTCTTTCTG TGCCTTTTGC GATTCTGCTT TAGTGCCACC

AGAAGATACT ACCTGGGTGC

781 AGTGGAACTG TCATGGGACT ATATGCAAAG TGATCTCGGT

GAGCTGCCTG TGGACGCAAG

841 ATTTCCTCCT AGAGTGCCAA AATCTTTTCC ATTCAACACC

TCAGTCGTGT ACAAAAAGAC

901 TCTGTTTGTA GAATTCACGG ATCACCTTTT CAACATCGCT

AAGCCAAGGC CACCCTGGAT

961 GGGTCTGCTA GGTCCTACCA TCCAGGCTGA GGTTTATGAT

ACAGTGGTCA TTACACTTAA

1021 GAACATGGCT TCCCATCCTG TCAGTCTTCA TGCTGTTGGT

GTATCCTACT GGAAAGCTTC

1081 TGAGGGAGCT GAATATGATG ATCAGACCAG TCAAAGGGAG

AAAGAAGATG ATAAAGTCTT

1141 CCCTGGTGGA AGCCATACAT ATGTCTGGCA GGTCCTGAAA

GAGAATGGTC CAATGGCCTC

1.201 TGACCCACTG TGCCTTACCT ACTCATATCT TTCTCATGTG

GACCTGGTAA AAGACTTGAA

1261 TTCAGGCCTC ATTGGAGCCC TACTAGTATG TAGAGAAGGG

AGTCTGGCCA AGGAAAAGAC

1321 ACAGACCTTG CACAAATTTA TACTACTTTT TGCTGTATTT

GATGAAGGGA AAAGTTGGCA

1381 CTCAGAAACA AAGAACTCCT TGATGCAGGA TAGGGATGCT

GCATCTGCTC GGGCCTGGCC

1441 TAAAATGCAC ACAGTCAATG GTTATGTAAA CAGGTCTCTG

CCAGGTCTGA TTGGATGCCA

1501 CAGGAAATCA GTCTATTGGC ATGTGATTGG AATGGGCACC

ACTCCTGAAG TGCACTCAAT 1561 ATTCCTCGAA GGTCACACAT TTCTTGTGAG GAACCATCGC

CAGGCGTCCT TGGAAATCTC

1621 GCCAATAACT TTCCTTACTG CTCAAACACT CTTGATGGAC

CTTGGACAGT TTCTACTGTT

1681 TTGTCATATC TCTTCCCACC AACATGATGG CATGGAAGCT

TATGTCAAAG TAGACAGCTG

1741 TCCAGAGGAA CCCCAACTAC GAATGAAAAA TAATGAAGAA

GCGGAAGACT ATGATGATGA

1801 TCTTACTGAT TCTGAAATGG ATGTGGTCAG GTTTGATGAT

GACAACTCTC CTTCCTTTAT

1861 CCAAATTCGC TCAGTTGCCA AGAAGCATCC TAAAACTTGG

GTACATTACA TTGCTGCTGA

1921 AGAGGAGGAC TGGGACTATG CTCCCTTAGT CCTCGCCCCC

GATGACAGAA GTTATAAAAG

1981 TCAATATTTG AACAATGGCC CTCAGCGGAT TGGTAGGAAG

TACAAAAAAG TCCGATTTAT

2041 GGCATACACA GATGAAACCT TTAAGACTCG TGAAGCTATT

CAGCATGAAT CAGGAATCTT

2101 GGGACCTTTA CTTTATGGGG AAGTTGGAGA CACACTGTTG

ATTATATTTA AGAATCAAGC

2161 AAGCAGACCA TATAACATCT ACCCTCACGG AATCACTGAT

GTCCGTCCTT TGTATTCAAG

2221 GAGATTACCA AAAGGTGTAA AACATTTGAA GGATTTTCCA

ATTCTGCCAG GAGAAATATT

2281 CAAATATAAA TGGACAGTGA CTGTAGAAGA TGGGCCAACT

AAATCAGATC CTCGGTGCCT

2341 GACCCGCTAT TACTCTAGTT TCGTTAATAT GGAGAGAGAT

CTAGCTTCAG GACTCATTGG

2401 CCCTCTCCTC ATCTGCTACA AAGAATCTGT AGATCAAAGA

GGAAACCAGA TAATGTCAGA

2461 CAAGAGGAAT GTCATCCTGT TTTCTGTATT TGATGAGAAC

CGAAGCTGGT ACCTCACAGA

2521 GAATATACAA CGCTTTCTCC CCAATCCAGC TGGAGTGCAG

CTTGAGGATC CAGAGTTCCA

2581 AGCCTCCAAC ATCATGCACA GCATCAATGG CTATGTTTTT

GATAGTTTGC AGTTGTCAGT

2641 TTGTTTGCAT GAGGTGGCAT ACTGGTACAT TCTAAGCATT

GGAGCACAGA CTGACTTCCT

2701 TTCTGTCTTC TTCTCTGGAT ATACCTTCAA ACACAAAATG

GTCTATGAAG ACACACTCAC

2761 CCTATTCCCA TTCTCAGGAG AAACTGTCTT CATGTCGATG

GAAAACCCAG GTCTATGGAT

2821 TCTGGGGTGC CACAACTCAG ACTTTCGGAA CAGAGGCATG

ACCGCCTTAC TGAAGGTTTC

2881 TAGTTGTGAC AAGAACACTG GTGATTATTA CGAGGACAGT

TATGAAGATA TTTCAGCATA

2941 CTTGCTGAGT AAAAACAATG CCATTGAACC AAGAAGCTTC

TCCCAGAATT CAAGACACCC

3001 TAGCACTAGG CAAAAGCAAT TTAATGCCAC CACAATTCCA

GAAAATGACA TAGAGAAGAC 3061 TGACCCTTGG TTTGCACACA GAACACCTAT GCCTAAAATA

CAAAATGTCT CCTCTAGTGA

3121 TTTGTTGATG CTCTTGCGAC AGAGTCCTAC TCCACATGGG

CTATCCTTAT CTGATCTCCA

3181 AGAAGCCAAA TATGAGACTT TTTCTGATGA TCCATCACCT

GGAGCAATAG ACAGTAATAA

3241 CAGCCTGTCT GAAATGACAC ACTTCAGGCC ACAGCTCCAT

CACAGTGGGG ACATGGTATT

3301 TACCCCTGAG TCAGGCCTCC AATTAAGATT AAA GAGAAA

CTGGGGACAA CTGCAGCAAC

3361 AGAGTTGAAG AAACTTGATT TCAAAGTTTC TAGTACATCA

AATAATCTGA TTTCAACAAT

3421 TCCATCAGAC AATTTGGCAG CAGGTACTGA TAATACAAGT TCCTTAGGAC CCCCAAGTAT

3481 GCCAGTTCAT TATGATAGTC AATTAGATAC CACTCTATTT

GGCAAAAAGT CATCTCCCCT

3541 TACTGAGTCT GGTGGACCTC TGAGCTTGAG TGAAGAAAAT

AATGATTCAA AGTTGTTAGA

3601 ATCAGGTTTA ATGAATAGCC AAGAAAGTTC ATGGGGAAAA

AATGTATCGT CAACAGAGAG

3661 TGGTAGGTTA TTTAAAGGGA AAAGAGCTCA TGGACCTGCT

TTGTTGACTA AAGATAATGC

3721 CTTATTCAAA GTTAGCATCT CTTTGTTAAA GACAAACAAA

ACTTCCAATA ATTCAGCAAC

3781 TAATAGAAAG ACTCACATTG ATGGCCCATC ATTATTAATT

GAGAATAGTC CATCAGTCTG

3841 GCAAAATATA TTAGAAAGTG ACACTGAGTT TAAAAAAGTG

ACACCTTTGA TTCATGACAG

3901 AATGCTTATG GACAAAAATG CTACAGCTTT GAGGCTAAAT

CATATGTCAA ATAAAACTAC

3961 TTCATCAAAA AACATGGAAA TGGTCCAACA GAAAAAAGAG

GGCCCCATTC CACCAGATGC

4021 ACAAAATCCA GATATGTCGT TCTTTAAGAT GCTATTCTTG

CCAGAATCAG CAAGGTGGAT

4081 ACAAAGGACT CATGGAAAGA ACTCTCTGAA CTCTGGGCAA

GGCCCCAGTC CAAAGCAATT

4141 AGTATCCTTA GGACCAGAAA AATCTGTGGA AGGTCAGAAT

TTCTTGTCTG AGAAAAACAA

4201 AGTGGTAGTA GGAAAGGGTG AATTTACAAA GGACGTAGGA

CTCAAAGAGA TGGTTTTTCC

4261 AAGCAGCAGA AACCTATTTC TTACTAACTT GGATAATTTA

CATGAAAATA ATACACACAA

4321 TCAAGAAAAA AAAATTCAGG AAGAAATAGA AAAGAAGGAA

ACATTAATCC AAGAGAATGT

4381 AGTTTTGCCT CAGATACATA CAGTGACTGG CACTAAGAAT

TTCATGAAGA ACCTTTTCTT

4441 ACTGAGCACT AGGCAAAATG TAGAAGGTTC ATATGACGGG

GCATATGCTC CAGTACTTCA

4501 AGATTTTAGG TCATTAAATG ATTCAACAAA TAGAACAAAG

AAACACACAG CTCATTTCTC 4561 AAAAAAAGGG GAGGAAGAAA ACTTGGAAGG CTTGGGAAAT

CAAACCAAGC AAATTGTAGA

4621 GAAATATGCA TGCACCACAA GGATATCTCC TAATACAAGC

CAGCAGAATT TTGTCACGCA

4681 ACGTAGTAAG AGAGCTTTGA AACAATTCAG ACTCCCACTA

GAAGAAACAG AACTTGAAAA

4741 AAGGATAATT GTGGATGACA CCTCAACCCA GTGGTCCAAA

AACATGAAAC ATTTGACCCC

4801 GAGCACCCTC ACACAGATAG ACTACAATGA GAAGGAGAAA

GGGGCCATTA CTCAGTCTCC

4861 CTTATCAGAT TGCCTTACGA GGAGTCATAG CATCCCTCAA

GCAAATAGAT CTCCATTACC

4921 CATTGCAAAG GTATCATCAT TTCCATCTAT TAGACCTATA

TATCTGACCA GGGTCCTATT

4981 CCAAGACAAC TCTTCTCATC TTCCAGCAGC ATCTTATAGA

AAGAAAGATT CTGGGGTCCA

5041 AGAAAGCAGT CATTTCTTAC AAGGAGCCAA AAAAAATAAC

CTTTCTTTAG CCATTCTAAC

5101 CTTGGAGATG ACTGGTGATC AAAGAGAGGT TGGCTCCCTG

GGGACAAGTG CCACAAATTC

5161 AGTCACATAC AAGAAAGTTG AGAACACTGT TCTCCCGAAA

CCAGACTTGC CCAAAACATC

5221 TGGCAAAGTT GAATTGCTTC CAAAAGTTCA CATTTATCAG

AAGGACCTAT TCCCTACGGA

5281 AACTAGCAAT GGGTCTCCTG GCCATCTGGA TCTCGTGGAA

GGGAGCCTTC TTCAGGGAAC

5341 AGAGGGAGCG ATTAAGTGGA ATGAAGCAAA CAGACCTGGA

AAAGTTCCCT TTCTGAGAGT

5401 AGCAACAGAA AGCTCTGCAA AGACTCCCTC CAAGCTATTG

GATCCTCTTG CTTGGGATAA

5461 CCACTATGGT ACTCAGATAC CAAAAGAAGA GTGGAAATCC

CAAGAGAAGT CACCAGAAAA

5521 AACAGCTTTT AAGAAAAAGG ATACCATTTT GTCCCTGAAC GCTTGTGAAA GCAATCATGC

5581 AATAGCAGCA ATAAATGAGG GACAAAATAA GCCCGAAATA

GAAGTCACCT GGGCAAAGCA

5641 AGGTAGGACT GAAAGGCTGT GCTCTCAAAA CCCACCAGTC

TTGAAACGCC ATCAACGGGA

5701 AATAACTCGT ACTACTCTTC AGTCAGATCA AGAGGAAATT GACTATGATG ATACCATATC

5761 AGTTGAAATG AAGAAGGAAG ATTTTGACAT TTATGATGAG

GATGAAAATC AGAGCCCCCG

5821 CAGCTTTCAA AAGAAAACAC GACACTATTT TATTGCTGCA GTGGAGAGGC TCTGGGATTA

5881 TGGGATGAGT AGCTCCCCAC ATGTTCTAAG AAACAGGGCT

CAGAGTGGCA GTGTCCCTCA

5941 GTTCAAGAAA GTTGTTTTCC AGGAATTTAC TGATGGCTCC

TTTACTCAGC CCTTATACCG

6001 TGGAGAACTA AATGAACATT TGGGACTCCT GGGGCCATAT

ATAAGAGCAG AAGTTGAAGA 6061 TAATATCATG GTAACTTTCA GAAATCAGGC CTCTCGTCCC

TATTCCTTCT ATTCTAGCCT

6121 TATTTCTTAT GAGGAAGATC AGAGGCAAGG AGCAGAACCT

AGAAAAAACT TTGTCAAGCC

6181 TAATGAAACC AAAACTTACT TTTGGAAAGT GCAACATCAT

ATGGCACCCA CTAAAGATGA

6241 GTTTGACTGC AAAGCCTGGG CTTATTTCTC TGATGTTGAC

CTGGAAAAAG ATGTGCACTC

6301 AGGCCTGATT GGACCCCTTC TGGTCTGCCA CACTAACACA

CTGAACCCTG CTCATGGGAG

6361 ACAAGTGACA GTACAGGAAT TTGCTCTGTT TTTCACCATC

TTTGATGAGA CCAAAAGCTG

6421 GTACTTCACT GAAAATATGG AAAGAAACTG CAGGGCTCCC TGCAATATCC AGATGGAAGA

6481 TCCCACTTTT AAAGAGAATT ATCGCTTCCA TGCAATCAAT

GGCTACATAA TGGATACACT

6541 ACCTGGCTTA GTAATGGCTC AGGATCAAAG GATTCGATGG

TATCTGCTCA GCATGGGCAG

6601 CAATGAAAAC ATCCATTCTA TTCATTTCAG TGGACATGTG

TTCACTGTAC GAAAAAAAGA

6661 GGAGTATAAA ATGGCACTGT ACAATCTCTA TCCAGGTGTT

TTTGAGACAG TGGAAATGTT

6721 ACCA CCAAA GCTGGAATTT GGCGGGTGGA ATGCCTTATT

GGCGAGCATC TACATGCTGG

6781 GATGAGCACA CTTTTTCTGG TGTACAGCAA TAAGTGTCAG

ACTCCCCTGG GAATGGCTTC

6841 TGGACACATT AGAGATTTTC AGATTACAGC TTCAGGACAA

TATGGACAGT GGGCCCCAAA

6901 GCTGGCCAGA CTTCATTATT CCGGATCAAT CAATGCCTGG AGCACCAAGG AGCCCTTTTC

6961 TTGGATCAAG GTGGATCTGT TGGCACCAAT GATTATTCAC

GGCATCAAGA CCCAGGGTGC

7021 CCGTCAGAAG TTCTCCAGCC TCTACATCTC TCAGTTTATC

ATCATGTATA GTCTTGATGG

7081 GAAGAAGTGG CAGACTTATC GAGGAAATTC CACTGGAACC

TTAATGGTCT TCTTTGGCAA

7141 TGTGGATTCA TCTGGGATAA AACACAATAT TTTTAACCCT

CCAATTATTG CTCGATACAT

7201 CCGTTTGCAC CCAACTCATT ATAGCATTCG CAGCACTCTT

CGCATGGAGT TGATGGGCTG

7261 TGATTTAAAT AGTTGCAGCA TGCCATTGGG AATGGAGAGT

AAAGCAATAT CAGATGCACA

7321 GATTACTGCT TCATCCTACT TTACCAATAT GTTTGCCACC

TGGTCTCCTT CAAAAGCTCG

7381 ACTTCACCTC CAAGGGAGGA GTAATGCCTG GAGACCTCAG

GTGAATAATC CAAAAGAGTG

7441 GCTGCAAGTG GACTTCCAGA AGACAATGAA AGTCACAGGA

GTAACTACTC AGGGAGTAAA

7501 ATCTCTGCTT ACCAGCATGT ATGTGAAGGA GTTCCTCATC

TCCAGCAGTC AAGATGGCCA 7 5 61 TCAGTGGACT CTCT T T T T TC AGAATGGCAA AGTAAAGGT T

T T TCAGGGAA A CAAGACTC

7 62 1 CT TCACACCT GTGGTGAACT CTCTAGACCC ACCGT TACTG

ACTCGCTACC T TCGAAT TCA

7 68 1 CCCCCAGAGT TGGGTGCACC AGAT TGCCCT GAGGATGGAG

GT TCTGGGCT GCGAGGCACA

7 7 4 1 GGACCTCTAC

*The underlined nucleic acids encode a signal peptide.

[0171] FVIII polypeptides include full-length FVIII, full-length FVIII minus Met at the N-terminus, mature FVIII (minus the signal sequence), mature FVIII with an additional Met at the N-terminus, and/or FVIII with a full or partial deletion of the B domain. In certain embodiments, FVIII variants include B domain deletions, whether partial or full deletions.

[0172] The sequence of native mature human FVIII is presented as SEQ ID NO:

4. A native FVIII protein has the following formula: Al-al-A2-a2-B-a3-A3-Cl- C2, where Al, A2, and A3 are the structurally-related "A domains," B is the "B domain," CI and C2 are the structurally-related "C domains," and al, a2 and a3 are acidic spacer regions. Referring to the primary amino acid sequence position in SEQ ID NO:4, the Al domain of human FVIII extends from Alal to about Arg336, the al spacer region extends from about Met337 to about Val374, the A2 domain extends from about Ala375 to about Tyr719, the a2 spacer region extends from about Glu720 to about Arg740, the B domain extends from about Ser741 to about Arg 1648, the a3 spacer region extends from about Glul649 to about Argl689, the A3 domain extends from about Serl690 to about Leu2025, the CI domain extends from about Gly2026 to about Asn2072, and the C2 domain extends from about Ser2073 to Tyr2332. Other than specific proteolytic cleavage sites, designation of the locations of the boundaries between the domains and regions of FVIII can vary in different literature references. The boundaries noted herein are therefore designated as approximate by use of the term "about."

[0173] The human FVIII gene was isolated and expressed in mammalian cells

(Toole, J. J., et al, Nature 312:342-347 (1984); Gitschier, J., et al, Nature 312:326-330 (1984); Wood, W. I., et al, Nature 312:330-337 (1984); Vehar, G. A., et al, Nature 312:337-342 (1984); WO 87/04187; WO 88/08035; WO 88/03558; and U.S. Pat. No. 4,757,006). The FVIII amino acid sequence was deduced from cDNA as shown in U.S. Pat. No. 4,965,199. In addition, partially or fully B-domain deleted FVIII is shown in U.S. Pat. Nos. 4,994,371 and 4,868,112. In some embodiments, the human FVIII B-domain is replaced with the human Factor V B-domain as shown in U.S. Pat. No. 5,004,803. The cDNA sequence encoding human Factor VIII and amino acid sequence are shown in SEQ ID NOs: 4 and 5, respectively, of US Application Publ. No. 2005/0100990.

[0174] The porcine FVIII sequence is published in Toole, J. J., et al., Proc. Natl.

Acad. Sci. USA 83:5939-5942 (1986). Further, the complete porcine cDNA sequence obtained from PCR amplification of FVIII sequences from a pig spleen cDNA library has been reported in Healey, J. F., et al, Blood 88:4209-4214 (1996). Hybrid human/porcine FVIII having substitutions of all domains, all subunits, and specific amino acid sequences were disclosed in U.S. Pat. No.

5,364,771 by Lollar and Runge, and in WO 93/20093. More recently, the nucleotide and corresponding amino acid sequences of the Al and A2 domains of porcine FVIII and a chimeric FVIII with porcine Al and/or A2 domains substituted for the corresponding human domains were reported in WO 94/11503. U.S. Pat. No. 5,859,204, Lollar, J. S., also discloses the porcine cDNA and deduced amino acid sequences. U.S. Pat. No. 6,458,563 discloses a B-domain- deleted porcine FVIII.

[0175] U.S. Pat. No. 5,859,204 to Lollar, J. S. reports functional mutants of FVIII having reduced antigenicity and reduced immunoreactivity. U.S. Pat. No.

6,376,463 to Lollar, J. S. also reports mutants of FVIII having reduced

immunoreactivity. US Appl. Publ. No. 2005/0100990 to Saenko et al. reports functional mutations in the A2 domain of FVIII.

[0176] In one embodiment, the FVIII (or FVIII portion of a chimeric protein) may be at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a FVIII amino acid sequence of amino acids 1 to 1438 of SEQ ID NO: 6 or amino acids 1 to 2332 of SEQ ID NO: 4 (without a signal sequence) or a FVIII amino acid sequence of amino acids 1 to 19 of SEQ ID NO: 3 and 1 to 1438 of SEQ ID NO: 6 or amino acids 1 to 19 of SEQ ID NO: 3 and amino acids 1 to 2332 of SEQ ID NO: 4 (with a signal sequence), wherein the FVIII has a clotting activity, e.g. , activates Factor IX as a cofactor to convert Factor X to activated Factor X. The FVIII (or FVIII portion of a chimeric protein) may be identical to a FVIII amino acid sequence of amino acids 1 to 1438 of SEQ ID NO: 6 or amino acids 1 to 2332 of SEQ ID NO: 4 (without a signal sequence). The FVIII may further comprise a signal sequence.

[0177] The "B-domain" of FVIII, as used herein, is the same as the B-domain known in the art that is defined by internal amino acid sequence identity and sites of proteolytic cleavage, e.g., residues Ser741-Argl648 of full-length human FVIII. The other human FVIII domains are defined by the following amino acid residues: Al, residues Alal-Arg372; A2, residues Ser373-Arg740; A3, residues Serl690- Asn2019; CI, residues Lys2020-Asn2172; C2, residues Ser2173-Tyr2332. The A3-C1-C2 sequence includes residues Serl690-Tyr2332. The remaining sequence, residues Glul649-Argl689, is usually referred to as the a3 acidic region. The locations of the boundaries for all of the domains, including the B-domains, for porcine, mouse and canine FVIII are also known in the art. In one embodiment, the B domain of FVIII is deleted ("B-domain-deleted factor VIII" or "BDD

FVIII"). An example of a BDD FVIII is REFACTO^® (recombinant BDD FVIII), which has the same sequence as the Factor VIII portion of the sequence in Table 5. (BDD FVIII heavy chain is double underlined; B domain is italicized; and BDD FVIII light chain is in plain text). A nucleotide sequence encoding the amino acid sequence set forth in Table 5 (SEQ ID NO: 7) is shown in Table 6.

TABLE 5. Amino Acid Sequence of B-domain Deleted Factor VIII (BDD

FVIII)

BDD FVIII (SEQ ID NO: 6)

ATRRY LGAVELS DY^

DHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASE GA^YDDQ^

KHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGP LLICYKESVDQRGNQIMSDKRVILFSVFDENRSWYLTENIQRFLPNPAGVQLED

KMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNT GDYYEDSYEDISAYLLSKNAIEPRS S F V PVL?; NXJJ¾EITR^TLOSDG^IDY

DD I sVEMKKE^"D^"FDI^"Y ^"6EDE QSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLR NRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVT FRNQASRPYSFYSSLI SYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDE FDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFF IFD ETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQR IRWYLLSMGSNE IHS IHFSGHVFTVRKKEEYKMALYNLYPGVFE VEMLPSKAG IWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPK LARLHYSGS INAWS KEPFSWIKVDLLAPMI IHGIKTQGARQKFSSLYI SQFI IM YSLDGKKWQTYRGNS GTLMVFFGNVDSSGIKHNIFNPPI IARYIRLHPTHYS IR S LRMELMGCDLNSCSMPLGMESKAI SDAQI ASSYFTNMFATWSPSKARLHLQG RSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLI SSSQDGH QWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVL GCEAQDLY

TABLE 6. Nucleotide Sequence Encoding BDD FVIII (SEQ ID NO: 7)*

661 A TGCAAATAGA

GCTCTCCACC TGCTTCTTTC

721 TGTGCCTTTT GCGATTCTGC TTTAGTGCCA CCAGAAGATA

CTACCTGGGT GCAGTGGAAC

781 TGTCATGGGA CTATATGCAA AGTGATCTCG GTGAGCTGCC

TGTGGACGCA AGATTTCCTC

841 CTAGAGTGCC AAAATCTTTT CCATTCAACA CCTCAGTCGT

GTACAAAAAG ACTCTGTTTG

901 TAGAATTCAC GGATCACCTT TTCAACATCG CTAAGCCAAG

GCCACCCTGG ATGGGTCTGC

961 TAGGTCCTAC CATCCAGGCT GAGGTTTATG ATACAGTGGT

CA ACAC T AAGAACA GG

1021 CTTCCCATCC TGTCAGTCTT CATGCTGTTG GTGTATCCTA

CTGGAAAGCT TCTGAGGGAG

1081 CTGAATATGA TGATCAGACC AGTCAAAGGG AGAAAGAAGA

TGATAAAGTC TTCCCTGGTG

1141 GAAGCCATAC ATATGTCTGG CAGGTCCTGA AAGAGAATGG

TCCAATGGCC TCTGACCCAC

1201 TGTGCCTTAC CTACTCATAT CTTTCTCATG TGGACCTGGT

AAAAGACTTG AATTCAGGCC

1261 TCATTGGAGC CCTACTAGTA TGTAGAGAAG GGAGTCTGGC

CAAGGAAAAG ACACAGACCT

1321 TGCACAAATT TATACTACTT TTTGCTGTAT TTGATGAAGG

GAAAAGTTGG CACTCAGAAA

1381 CAAAGAACTC CTTGATGCAG GA AGGGATG CTGCA CTGC

TCGGGCCTGG CCTAAAATGC

1441 ACACAGTCAA TGGTTATGTA AACAGGTCTC TGCCAGGTCT

GA GGATGC CACAGGAAAT

1501 CAGTCTATTG GCATGTGATT GGAATGGGCA CCACTCCTGA

AGTGCACTCA ATATTCCTCG

1561 AAGGTCACAC ATTTCTTGTG AGGAACCATC GCCAGGCGTC

CTTGGAAATC TCGCCAATAA

1621 CTTTCCTTAC TGCTCAAACA CTCTTGATGG ACCTTGGACA

GTTTCTACTG TTTTGTCATA 1681 TCTCTTCCCA CCAACATGAT GGCATGGAAG CTTATGTCAA

AGTAGACAGC TGTCCAGAGG

1741 AACCCCAACT ACGAATGAAA AATAATGAAG AAGCGGAAGA

CTATGATGAT GATCTTACTG

1801 ATTCTGAAAT GGATGTGGTC AGGTTTGATG ATGACAACTC

TCCTTCCTTT ATCCAAATTC

1861 GCTCAGTTGC CAAGAAGCAT CCTAAAACTT GGGTACATTA

CATTGCTGCT GAAGAGGAGG

1921 ACTGGGACTA TGCTCCCTTA GTCCTCGCCC CCGATGACAG

AAGTTATAAA AGTCAATATT

1981 TGAACAATGG CCCTCAGCGG ATTGGTAGGA AGTACAAAAA

AGTCCGATTT ATGGCATACA

2041 CAGATGAAAC CTTTAAGACT CGTGAAGCTA TTCAGCATGA

ATCAGGAATC TTGGGACCTT

2101 TACTTTATGG GGAAGTTGGA GACACACTGT TGATTATATT

TAAGAATCAA GCAAGCAGAC

2161 CATATAACAT CTACCCTCAC GGAATCACTG ATGTCCGTCC

TTTGTATTCA AGGAGATTAC

2221 CAAAAGGTGT AAAACATTTG AAGGATTTTC CAATTCTGCC

AGGAGAAA A TTCAAA A A

2281 AATGGACAGT GACTGTAGAA GATGGGCCAA CTAAATCAGA

TCCTCGGTGC CTGACCCGCT

2341 ATTACTCTAG TTTCGTTAAT ATGGAGAGAG ATCTAGCTTC

AGGACTCATT GGCCCTCTCC

2401 TCATCTGCTA CAAAGAATCT GTAGATCAAA GAGGAAACCA

GATAATGTCA GACAAGAGGA

2461 ATGTCATCCT GTTTTCTGTA TTTGATGAGA ACCGAAGCTG

GTACCTCACA GAGAATATAC

2521 AACGCTTTCT CCCCAATCCA GCTGGAGTGC AGCTTGAGGA

TCCAGAGTTC CAAGCCTCCA

2581 ACATCATGCA CAGCATCAAT GGCTATGTTT TTGATAGTTT

GCAGTTGTCA GTTTGTTTGC

2641 ATGAGGTGGC ATACTGGTAC ATTCTAAGCA TTGGAGCACA

GACTGACTTC CTTTCTGTCT

2701 TCTTCTCTGG ATATACCTTC AAACACAAAA TGGTCTATGA

AGACACACTC ACCCTATTCC

2761 CATTCTCAGG AGAAACTGTC TTCATGTCGA TGGAAAACCC

AGGTCTATGG ATTCTGGGGT

2821 GCCACAACTC AGACTTTCGG AACAGAGGCA TGACCGCCTT

ACTGAAGGTT TCTAGTTGTG

2881 ACAAGAACAC TGGTGATTAT TACGAGGACA GTTATGAAGA

TATTTCAGCA TACTTGCTGA

2941 GTAAAAACAA TGCCATTGAA CCAAGAAGCT TCTCTCAAAA

CCCACCAGTC TTGAAACGCC

3001 ATCAACGGGA AATAACTCGT ACTACTCTTC AGTCAGATCA

AGAGGAAATT GACTATGATG

3061 ATACCATATC AGTTGAAATG AAGAAGGAAG ATTTTGACAT

TTATGATGAG GATGAAAATC

3121 AGAGCCCCCG CAGCTTTCAA AAGAAAACAC GACACTATTT

TATTGCTGCA GTGGAGAGGC 3181 TCTGGGATTA TGGGATGAGT AGCTCCCCAC ATGTTCTAAG AAACAGGGCT CAGAGTGGCA

3241 GTGTCCCTCA GTTCAAGAAA GTTGTTTTCC AGGAATTTAC

TGATGGCTCC TTTACTCAGC

3301 CCTTATACCG TGGAGAACTA AATGAACATT TGGGACTCCT

GGGGCCATAT ATAAGAGCAG

3361 AAGTTGAAGA TAATATCATG GTAACTTTCA GAAATCAGGC

CTCTCGTCCC TATTCCTTCT

3421 ATTCTAGCCT TATTTCTTAT GAGGAAGATC AGAGGCAAGG AGCAGAACCT AGAAAAAACT

3481 TTGTCAAGCC TAATGAAACC AAAACTTACT TTTGGAAAGT

GCAACATCAT ATGGCACCCA

3541 CTAAAGATGA GTTTGACTGC AAAGCCTGGG CTTATTTCTC

TGATGTTGAC CTGGAAAAAG

3601 ATGTGCACTC AGGCCTGATT GGACCCCTTC TGGTCTGCCA

CACTAACACA CTGAACCCTG

3661 CTCATGGGAG ACAAGTGACA GTACAGGAAT TTGCTCTGTT

TTTCACCATC TTTGATGAGA

3721 CCAAAAGCTG GTACTTCACT GAAAATATGG AAAGAAACTG

CAGGGCTCCC TGCAATATCC

3781 AGATGGAAGA TCCCACTTTT AAAGAGAATT ATCGCTTCCA

TGCAATCAAT GGCTACATAA

3841 TGGATACACT ACCTGGCTTA GTAATGGCTC AGGATCAAAG

GATTCGATGG TATCTGCTCA

3901 GCATGGGCAG CAATGAAAAC ATCCATTCTA TTCATTTCAG

TGGACATGTG TTCACTGTAC

3961 GAAAAAAAGA GGAGTATAAA ATGGCACTGT ACAATCTCTA

TCCAGGTGTT TTTGAGACAG

4021 TGGAAATGTT ACCATCCAAA GCTGGAATTT GGCGGGTGGA

ATGCCTTATT GGCGAGCATC

4081 TACATGCTGG GATGAGCACA CTTTTTCTGG TGTACAGCAA

TAAGTGTCAG ACTCCCCTGG

4141 GAATGGCTTC TGGACACATT AGAGATTTTC AGATTACAGC

TTCAGGACAA TATGGACAGT

4201 GGGCCCCAAA GCTGGCCAGA CTTCATTATT CCGGATCAAT

CAATGCCTGG AGCACCAAGG

4261 AGCCCTTTTC TTGGATCAAG GTGGATCTGT TGGCACCAAT

GATTATTCAC GGCATCAAGA

4321 CCCAGGGTGC CCGTCAGAAG TTCTCCAGCC TCTACATCTC TCAGTTTATC ATCATGTATA

GTCTTGATGG GAAGAAGTGG CAGACTTATC GAGGAAATTC

CACTGGAACC TTAATGGTCT

4441 TCTTTGGCAA TGTGGATTCA TCTGGGATAA AACACAATAT

TTTTAACCCT CCAATTATTG

4501 CTCGATACAT CCGTTTGCAC CCAACTCATT ATAGCATTCG

CAGCACTCTT CGCATGGAGT

4561 TGATGGGCTG TGATTTAAAT AGTTGCAGCA TGCCATTGGG

AATGGAGAGT AAAGCAATAT

4621 CAGATGCACA GATTACTGCT TCATCCTACT TTACCAATAT

GTTTGCCACC TGGTCTCCTT 68 1 CAAAAGCTCG ACT TCACCTC CAAGGGAGGA GTAATGCCTG

GAGACCTCAG GTGAATAATC

7 4 1 CAAAAGAGTG GCTGCAAGTG GACT TCCAGA AGACAATGAA

AGTCACAGGA GTAACTACTC

8 0 1 AG G GAG AAA ATCTCTGCT T ACCAGCATGT ATGTGAAGGA

GT TCCTCATC TCCAGCAGTC

8 61 AAGATGGCCA TCAGTGGACT CTCT T T T T TC AGAATGGCAA

AGTAAAGGT T T T TCAGGGAA

92 1 ATCAAGACTC CT TCACACCT GTGGTGAACT CTCTAGACCC

ACCGT TACTG ACTCGCTACC

98 1 T TCGAAT TCA CCCCCAGAGT TGGGTGCACC AGAT TGCCCT

GAGGATGGAG GT TCTGGGCT

0 4 1 GCGAGGCACA GGACCTCTAC

*The underlined nucleic acids encode a signal peptide.

A "B-domain-deleted FVIII" may have the full or partial deletions disclosed in U.S. Pat. Nos. 6,316,226, 6,346,513, 7,041,635, 5,789,203, 6,060,447, 5,595,886, 6,228,620, 5,972,885, 6,048,720, 5,543,502, 5,610,278, 5,171,844, 5,112,950, 4,868,112, and 6,458,563. In some embodiments, a B-domain-deleted FVIII sequence of the present invention comprises any one of the deletions disclosed at col. 4, line 4 to col. 5, line 28 and Examples 1-5 of U.S. Pat. No. 6,316,226 (also in US 6,346,513). In another embodiment, a B-domain deleted Factor VIII is the S743/Q1638 B-domain deleted Factor VIII (SQ BDD FVIII) (e.g., Factor VIII having a deletion from amino acid 744 to amino acid 1637, e.g., Factor VIII having amino acids 1-743 and amino acids 1638-2332 of SEQ ID NO: 4, i.e., SEQ ID NO: 6). In some embodiments, a B-domain-deleted FVIII of the present invention has a deletion disclosed at col. 2, lines 26-51 and examples 5-8 of U.S. Patent No. 5,789,203 (also US 6,060,447, US 5,595,886, and US

6,228,620). In some embodiments, a B-domain-deleted Factor VIII has a deletion described in col. 1, lines 25 to col. 2, line 40 of US Patent No. 5,972,885; col. 6, lines 1-22 and example 1 of U.S. Patent no. 6,048,720; col. 2, lines 17-46 of U.S. Patent No. 5,543,502; col. 4, line 22 to col. 5, line 36 of U.S. Patent no. 5,171,844; col. 2, lines 55-68, figure 2, and example 1 of U.S. Patent No. 5,112,950; col. 2, line 2 to col. 19, line 21 and table 2 of U.S. Patent No. 4,868,112; col. 2, line 1 to col. 3, line 19, col. 3, line 40 to col. 4, line 67, col. 7, line 43 to col. 8, line 26, and col. 11, line 5 to col. 13, line 39 of U.S. Patent no. 7,041,635; or col. 4, lines 25- 53, of U.S. Patent No. 6,458,563. In some embodiments, a B-domain-deleted FVIII has a deletion of most of the B domain, but still contains amino -terminal sequences of the B domain that are essential for in vivo proteolytic processing of the primary translation product into two polypeptide chain, as disclosed in WO 91/09122. In some embodiments, a B-domain-deleted FVIII is constructed with a deletion of amino acids 747-1638, i.e., virtually a complete deletion of the B domain. Hoeben R.C., et al. J. Biol. Chem. 265 (13): 7318-7323 (1990). A B- domain-deleted Factor VIII may also contain a deletion of amino acids 771-1666 or amino acids 868-1562 of FVIII. Meulien P., et al. Protein Eng. 2(4): 301-6 (1988). Additional B domain deletions that are part of the invention include: deletion of amino acids 982 through 1562 or 760 through 1639 (Toole et al, Proc. Natl. Acad. Sci. U.S.A. (1986) 83, 5939-5942)), 797 through 1562 (Eaton, et al. Biochemistry (1986) 25:8343-8347)), 741 through 1646 (Kaufman (PCT published application No. WO 87/04187)), 747-1560 (Sarver, et al, DNA (1987) 6:553-564)), 741 through 1648 (Pasek (PCT application No.88/00831)), or 816 through 1598 or 741 through 1648 (Lagner (Behring Inst. Mitt. (1988) No 82: 16- 25, EP 295597)). In other embodiments, BDD FVIII includes a FVIII polypeptide containing fragments of the B-domain that retain one or more N-linked

glycosylation sites, e.g., residues 757, 784, 828, 900, 963, or optionally 943, which correspond to the amino acid sequence of the full-length FVIII sequence. Examples of the B-domain fragments include 226 amino acids or 163 amino acids of the B-domain as disclosed in Miao, H.Z., et al, Blood 103(a): 3412-3419 (2004), Kasuda, A, et al, J. Thromb. Haemost. 6: 1352-1359 (2008), and Pipe, S.W., et al, J. Thromb. Haemost. 9: 2235-2242 (2011) {i.e., the first 226 amino acids or 163 amino acids of the B domain are retained). In still other

embodiments, BDD FVIII further comprises a point mutation at residue 309 (from Phe to Ser) to improve expression of the BDD FVIII protein. See Miao, H.Z., et al, Blood 103(a): 3412-3419 (2004). In still other embodiments, the BDD FVIII includes a FVIII polypeptide containing a portion of the B-domain, but not containing one or more furin cleavage sites {e.g., Argl313 and Arg 1648). See Pipe, S.W., et al, J. Thromb. Haemost. 9: 2235-2242 (2011). Each of the foregoing deletions may be made in any FVIII sequence.

In some embodiments, the FVIII has a partial B-domain. In some embodiments, the FVIII protein with a partial B-domain is FVIII 198 (SEQ ID NO: 89). FVIII198 is a partial B-domain containing single chain FVIIIFc molecule-226N6. 226 represents the N-terminus 226 amino acid of the FVIII B- domain, and N6 represents six N-glycosylation sites in the B-domain.

[0180] In one embodiment, FVIII is cleaved right after arginine at amino acid

1648 (in full-length Factor VIII or SEQ ID NO: 4), amino acid 754 (in the

S743/Q1638 B-domain deleted Factor VIII or SEQ ID NO: 6), or the

corresponding arginine residue (in other variants), thereby resulting in a heavy chain and a light chain. In another embodiment, FVIII comprises a heavy chain and a light chain, which are linked or associated by a metal ion-mediated non- covalent bond.

[0181] In other embodiments, FVIII is a single chain FVIII that has not been

cleaved right after Arginine at amino acid 1648 (in full-length FVIII or SEQ ID NO: 4), amino acid 754 (in the S743/Q1638 B-domain-deleted FVIII or SEQ ID NO: 6), or the corresponding Arginine residue (in other variants). A single chain FVIII may comprise one or more amino acid substitutions. In one embodiment, the amino acid substitution is at a residue corresponding to residue 1648, residue 1645, or both of full-length mature Factor VIII polypeptide (SEQ ID NO: 4) or residue 754, residue 751, or both of SQ BDD Factor VIII (SEQ ID NO: 6). The amino acid substitution can be any amino acids other than arginine, e.g. , isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, selenocysteine, serine, tyrosine, histidine, ornithine, pyrrolysine, or taurine.

[0182] FVIII can further be cleaved by thrombin and then activated as FVIIIa, serving as a cofactor for activated Factor IX (FIXa). And the activated FIX together with activated FVIII forms a Xase complex and converts Factor X to activated Factor X (FXa). For activation, FVIII is cleaved by thrombin after three Arginine residues, at amino acids 372, 740, and 1689 (corresponding to amino acids 372, 740, and 795 in the B-domain deleted FVIII sequence), the cleavage generating FVIIIa having the 50kDa Al, 43kDa A2, and 73kDa A3-C1-C2 chains. In one embodiment, the FVIII protein useful for the present invention is non- active FVIII. In another embodiment, the FVIII protein is an activated FVIII.

[0183] The protein having FVIII polypeptide linked to or associated with the

VWF fragment can comprise a sequence at least 50%, 60%, 70%>, 80%>, 90%>, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4 or 6, wherein the sequence has the FVIII clotting activity, e.g., activating Factor IX as a cofactor to convert Factor X to activated Factor X (FXa).

[0184] "Hybrid" or "chimeric" polypeptides and proteins, as used herein, includes a combination of a first polypeptide chain, e.g. , the VWF fragment, optionally fused to a first Ig constant region or a portion thereof, with a second polypeptide chain, e.g., a FVIII protein linked to an XTEN sequence, optionally fused to a second Ig constant region or a portion thereof, thereby forming a heterodimer. In one embodiment, the first polypeptide and the second polypeptide in a hybrid are associated with each other via protein-protein interactions, such as charge-charge or hydrophobic interactions. In another embodiment, the first polypeptide and the second polypeptide in a hybrid are associated with each other via disulfide or other covalent bond(s). Hybrids are described, for example, in US 2004/101740 and US 2006/074199. The second polypeptide may be an identical copy of the first polypeptide or a non-identical polypeptide. In one embodiment, the first polypeptide is a FVIII protein(X)-Fc fusion protein, and the second polypeptide is a polypeptide comprising, consisting essentially of, or consisting of an Fc region, wherein the first polypeptide and the second polypeptide are associated with each other. In another embodiment, the first polypeptide comprises a VWF fragment- XTEN-Fc fusion protein, and the second polypeptide comprises FVIII-Fc fusion protein, making the hybrid a heterodimer. In other embodiments, the first polypeptide comprises a VWF fragment-Fc fusion protein, and the second polypeptide comprises FVIII(X)-Fc fusion protein, making the hybrid a heterodimer. In yet other embodiments, the first polypeptide comprises a VWF fragment-XTEN-Fc fusion protein, and the second polypeptide comprises FVIII(X)-Fc fusion protein. The first polypeptide and the second polypeptide can be associated through a covalent bond, e.g., a disulfide bond, between the first Fc region and the second Fc region. The first polypeptide and the second polypeptide can further be associated with each other by binding between the VWF fragment and the FVIII protein.

[0185] A FVIII protein useful in the present invention can include FVIII having one or more additional XTEN sequences, which do not affect the FVIII coagulation activity. Such XTEN sequences can be fused to the C-terminus or N- terminus of the FVIII protein or inserted between one or more of the two amino acid residues in the FVIII protein wherein the insertions do not affect the FVIII coagulation activity or FVIII function. In one embodiment, the insertions improve pharmacokinetic properties of the FVIII protein (e.g., half-life). In another embodiment, the insertions can be multiple insertions, e.g., more than two, three, four, five, six, seven, eight, nine, or ten insertions. Examples of the insertion sites include, but are not limited to, the sites listed in Tables 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 or any combinations thereof.

[0186] The FVIII protein linked to one or more XTEN sequences can be

represented as FVIII(X), FVIII(Xl), FVIII_fa→^-X-FVIII_{fc→rf ,} wherein FVlll_(a→b) comprises, consists essentially of, or consists of a first portion of a FVIII protein from amino acid residue "a" to amino acid residue "b"; X or XI comprises, consists essentially of, or consists of one or more XTEN sequences, FVUl(_c→d) comprises, consists essentially of, or consists of a second portion of a FVIII protein from amino acid residue "c" to amino acid residue "d";

a is the N-terminal amino acid residue of the first portion of the FVIII protein, b is the C-terminal amino acid residue of the first portion of the FVIII protein but is also the N-terminal amino acid residue of the two amino acids of an insertion site in which the XTEN sequence is inserted,

c is the N-terminal amino acid residue of the second portion of the FVIII protein but is also the C-terminal amino acid residue of the two amino acids of an insertion site in which the XTEN sequence is inserted, and

d is the C-terminal amino acid residue of the FVIII protein, and

wherein the first portion of the FVIII protein and the second portion of the FVIII protein are not identical to each other and are of sufficient length together such that the FVIII protein has a FVIII coagulation activity.

[0187] In one embodiment, the first portion of the FVIII protein and the second portion of the FVIII protein are fragments of SEQ ID NO: 4 [full length mature FVIII sequence] or SEQ ID NO: 6 [B-domain deleted FVIII], e.g., N-terminal portion and C-terminal portion, respectively. In certain embodiments, the first portion of the FVIII protein comprises the Al domain and the A2 domain of the FVIII protein. The second portion of the FVIII protein comprises the A3 domain, the CI domain, and optionally the C2 domain. In yet other embodiments, the first portion of the FVIII protein comprises the Al domain and A2 domain, and the second portion of the FVIII protein comprises a portion of the B domain, the A3 domain, the CI domain, and optionally the C2 domain. In still other

embodiments, the first portion of the FVIII protein comprises the Al domain, A2 domain, and a portion of the B domain of the FVIII protein, and the second portion of the FVIII protein comprises the A3 domain, the CI domain, and optionally the C2 domain. In still other embodiments, the first portion of the FVIII protein comprises the Al domain, A2 domain, and a first portion of the B domain of the FVIII protein. The second portion of the FVIII protein comprises a second portion of the B domain, the A3 domain, the C 1 domain, and optionally the C2 domain. In some embodiments, the two amino acids ("b" and "c") can be any one or more of the amino acid residues insertion sites shown in Tables 7, 8, 9, 10, 11, 12, 13, 14, and 15. For example, "b" can be the amino acid residue immediately upstream of the site in which one or more XTEN sequences are inserted or linked, and "c" can be the amino acid residue immediately downstream of the site in which the one or more XTEN sequences are inserted or linked. In some embodiments, "a" is the first mature amino acid sequence of a FVIII protein, and "d" is the last amino acid sequence of a FVIII protein. For example,

FVIII^i, can be an amino acid sequence at least 70%, 80%, 90%>, 95%>, 96%>, 97%, 98%, 99%, or 100% identical to amino acids 1 to 745 of SEQ ID NO: 6 [B domain deleted FVIII amino acid sequence] or SEQ ID NO: 4 [full length FVIII] and FVUl(_c→d) can be amino acids 746 to 1438 of SEQ ID NO: 6 or amino acids 1641 to 2332 of SEQ ID NO: 4, respectively.

In some aspects, the insertion site in the FVIII protein is located in one or more domains of the FVIII protein, which is the N-terminus, the Al domain, the A2 domain, the A3 domain, the B domain, the CI domain, the C2 domain, the C- terminus, or two or more combinations thereof or between two domains of the FVIII protein, which are the Al domain and al acidic region, and the al acidic region and A2 domain, the A2 domain and a2 acidic region, the a2 acidic region and B domain, the B domain and A3 domain, and the A3 domain and C 1 domain, the CI domain and C2 domain, or any combinations thereof. For example, the insertion sites in which the XTEN sequence can be inserted are selected from the group consisting of the N-terminus and Al domain, the N-terminus and A2 domain, the N-terminus and A3 domain, the N-terminus and B domain, the N- terminus and CI domain, the N-terminus and C2 domain, the N-terminus and the C -terminus, the Al and A2 domains, the Al and A3 domains, the Al and B domains, the Al and CI domains, the Al and C2 domains, the Al domain and the C-terminus, the A2 and A3 domains, the A2 and B domains, the A2 and CI domains, the A2 and C2 domains, the A2 domain and the C-terminus, the A3 and B domains, the A3 and C 1 domains, the A3 and C2 domains, the A3 domain and the C-terminus, the B and CI domains, the B and C2 domains, the B domain and the C-terminus, the CI and C2 domains, the CI and the C-terminus, the C2 domain, and the C-terminus, and two or more combinations thereof. Non-limiting examples of the insertion sites are listed in Tables 7, 8, 9, 10, 11, 12, 13, 14, and 15.

[0189] The FVIII protein, in which the XTEN sequence is inserted immediately downstream of one or more amino acids (e.g., one or more XTEN insertion sites) in the FVIII protein or linked at the C-terminus or the N-terminus, retains the FVIII activity after linkage to or insertion by the XTEN sequence. The XTEN sequence can be inserted in the FVIII protein once or more than once, twice, three times, four times, five times, or six times such that the insertions do not affect the FVIII activity (i.e., the FVIII protein still retains the coagulation property).

[0190] The FVIII protein useful in the present invention can be linked to one or more XTEN polypeptides at the N-terminus or C-terminus of the FVIII protein by an optional linker or inserted immediately downstream of one or more amino acids (e.g., one or more XTEN insertion sites) in the FVIII protein by one or more optional linkers. In one embodiment, the two amino acid residues in which the XTEN sequence is inserted or the amino acid residue to which the XTEN sequence is linked correspond to the two or one amino acid residues of SEQ ID NO: 4 [full length mature FVIII] selected from the group consisting of the residues in Table 7, Table 8, Table 9, and Table 10 and any combinations thereof.

[0191] In other embodiments, at least one XTEN sequence is inserted in any one or more XTEN insertion sites disclosed herein or any combinations thereof. In one aspect, at least one XTEN sequence is inserted in one or more XTEN insertion sites disclosed in one or more amino acids disclosed in Table 7. TABLE 7: Exemplary XTEN Insertion Sites

* Indicates an insertion point for XTEN based on the amino acid number of mature full- length human FVIII, wherein the insertion could be either on the N- or C-terminal side of the indicated amino acid.

192] In some embodiments, one or more XTEN sequences are inserted within about six amino acids up or down from amino acids 32, 220, 224, 336, 339, 399, 416, 603, 1656, 171 1 , 1725, 1905, or 1910, corresponding to SEQ ID NO: 4 or any combinations thereof.

TABLE 8. Exemplary XTEN Insertion Ranges

*Distance from insertion residue refers to the relative number of amino acids away from the N-terminus (negative numbers) or C-terminus (positive numbers) of the designated insertion residue (residue "0") where an insertion may be made. The designation "-x" refers to an insertion site which is x amino acids away on the N- terminal side of the designated insertion residue. Similarly, the designation "+x" refers to an insertion site which is x amino acids away on the C -terminal side of the designated insertion residue.

For example, "-1 , +2" indicates that the insertion is made at the N-terminus or C- terminus of amino acid residues denoted -1 , 0, +1 or +2.

[0193] In other embodiments, one or more XTEN sequences are inserted

immediately down stream of one or more amino acids corresponding to the full- length mature human FVIII selected from the group consisting of one or more insertion sites in Table 9.

TABLE 9. Exemplary XTEN Insertion Sites or Ranges

* indicates range of insertion sites numbered relative to the amino acid number of mature human FVIII

[0194] In yet other embodiments, one or more XTENs are inserted in the B

domain of FVIII. In one example, an XTEN is inserted between amino acids 740 and 1640 corresponding to SEQ ID NO: 4, wherein the FVIII sequence between amino acids 740 and 1640 is optionally not present. In another example, an XTEN is inserted between amino acids 741 and 1690 corresponding to SEQ ID NO: 4, wherein the FVIII sequence between amino acids 740 and 1690 is optionally not present. In other examples, an XTEN is inserted between amino acids 741 and 1648 corresponding to SEQ ID NO: 4, wherein the FVIII sequence between amino acids 741 and 1648 is optionally not present. In yet other examples, an XTEN is inserted between amino acids 743 and 1638 corresponding to SEQ ID NO: 4, wherein the FVIII sequence between amino acids 743 and 1638 is optionally not present. In still other examples, an XTEN is inserted between amino acids 745 and 1656 corresponding to SEQ ID NO: 4, wherein the FVIII sequence between amino acids 745 and 1656 is optionally not present. In some examples, an XTEN is inserted between amino acids 745 and 1657 corresponding to SEQ ID NO: 4, wherein the FVIII sequence between amino acids 745 and 1657 is optionally not present. In certain examples, an XTEN is inserted between amino acids 745 and 1667 corresponding to SEQ ID NO: 4, wherein the FVIII sequence between amino acids 745 and 1667 is optionally not present. In still other examples, an XTEN is inserted between amino acids 745 and 1686 corresponding to SEQ ID NO: 4, wherein the FVIII sequence between amino acids 745 and 1686 is optionally not present. In some other examples, an XTEN is inserted between amino acids 747 and 1642 corresponding to SEQ ID NO: 4, wherein the FVIII sequence between amino acids 747 and 1642 is optionally not present. In still other examples, an XTEN is inserted between amino acids 751 and 1667 corresponding to SEQ ID NO: 4, wherein the FVIII sequence between amino acids 751 and 1667 is optionally not present.

In some embodiments, one or more XTENs are inserted in one or more amino acids immediately downstream of an amino acid of an insertion site selected from the group consisting of the amino acid residues in Table 10.

- I l l - TABLE 10: FVIII XTEN insertion sites and construct designations

* Indicates the amino acid number of the mature FVIII protein In one embodiment, the one or more XTEN insertion sites are located within one or more surface-exposed, flexible loop structure of the FVIII protein (e.g., a permissive loop). For example, at least one XTEN sequence can be inserted in each FVIII "A" domain comprising at least two "permissive loops" into which at least one XTEN polypeptide can be inserted without eliminating procoagulant activity of the recombinant protein, or the ability of the recombinant proteins to be expressed in vivo or in vitro in a host cell. The permissive loops are regions that allow insertion of at least one XTEN sequence with, among other attributes, high surface or solvent exposure and high conformational flexibility. The Al domain comprises a permissive loop-1 (Al-1) region and a permissive loop-2 (A 1-2) region, the A2 domain comprises a permissive loop-1 (A2-1) region and a permissive loop-2 (A2-2) region, the A3 domain comprises a permissive loop-1 (A3-1) region and a permissive loop-2 (A3 -2) region. [0197] In one aspect, a first permissive loop in the FVIII Al domain (Al-1) is located between beta strand 1 and beta strand 2, and a second permissive loop in the FVIII A2 domain (Al-2) is located between beta strand 11 and beta strand 12. A first permissive loop in the FVIII A2 domain (A2-1) is located between beta strand 22 and beta strand 23, and a second permissive loop in the FVIII A2 domain (A2-2) is located between beta strand 32 and beta strand 33. A first permissive loop in the FVIII A3 domain (A3-1) is located between beta strand 38 and beta strand 39, and a second permissive loop in the FVIII A3 (A3 -2) is located between beta strand 45 and beta strand 46. In certain aspects, the surface-exposed, flexible loop structure comprising Al-1 corresponds to a region in native mature human FVIII from about amino acid 15 to about amino acid 45 of SEQ IDNO: 4, e.g., from about amino acid 18 to about amino acid 41 of SEQ ID NO: 4. In other aspects, the surface-exposed, flexible loop structure comprising Al-2 corresponds to a region in native mature human FVIII from about amino acid 201 to about amino acid 232 of SEQ ID NO: 4, e.g., from about amino acid 218 to about amino acid 229 of SEQ ID NO: 4. In yet other aspects, the surface-exposed, flexible loop structure comprising A2-1 corresponds to a region in native mature human FVIII from about amino acid 395 to about amino acid 421 of SEQ ID NO: 4, e.g. from about amino acid 397 to about amino acid 418 of SEQ ID NO: 4. In still other embodiments, the surface-exposed, flexible loop structure comprising A2-2 corresponds to a region in native mature human FVIII from about amino acid 577 to about amino acid 635 of SEQ ID NO: 4, e.g., from about amino acid 595 to about amino acid 607 of SEQ ID NO: 4. In certain aspects the surface-exposed, flexible loop structure comprising A3-1 corresponds to a region in native mature human FVIII from about amino acid 1705 to about amino acid 1732 of SEQ ID NO: 4, e.g., from about amino acid 1711 to about amino acid 1725 of SEQ ID NO: 4. In yet other aspects, the surface-exposed, flexible loop structure comprising A3 -2 corresponds to a region in native mature human FVIII from about amino acid 1884 to about amino acid 1917 of SEQ ID NO: 4, e.g., from about amino acid 1899 to about amino acid 1911 of SEQ ID NO: 4.

[0198] In another embodiment, the one or more amino acids in which at least one

XTEN sequence is inserted is located within a3 domain, e.g., amino acids 1649 to 1689, corresponding to full-length mature FVIII polypeptide. In a particular embodiment, an XTEN sequence is inserted between amino acids 1656 and 1657 of SEQ ID NO: 4 (full-length mature FVIII). In a specific embodiment, a FVIII protein comprising an XTEN sequence inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO: 4 further comprises a deletion from amino acid 745 to amino acid 1656 corresponding to SEQ ID NO: 4.

In some embodiments, the one or more insertion sites for one or more

XTEN insertions are immediately downstream of one or more amino acids selected from the group consisting of:

(1) amino acid 3, (2) amino acid 18, (3) amino acid 22, (4) amino acid 26, (5) amino acid 32, (6) amino acid 40, (7) amino acid 60, (8) amino acid 65, (9) amino acid 81 , (10) amino acid 116, (11) amino acid 119, (12) amino acid 130, (13) amino acid 188, (14) amino acid 211, (15) amino acid 216, (16) amino acid 220, (17) amino acid 224, (18) amino acid 230, (19) amino acid 333, (20) amino acid 336, (21) amino acid 339, (22) amino acid 375, (23) amino acid 399, (24) amino acid 403, (25) amino acid 409, (26) amino acid 416, (26) amino acid 442, (28) amino acid 487, (29) amino acid 490, (30) amino acid 494, (31) amino acid 500, (32) amino acid 518, (33) amino acid 599, (34) amino acid 603, (35) amino acid 713, (36) amino acid 745, (37) amino acid 1656, (38) amino acid 1711, (39) amino acid 1720,

(40) amino acid 1725, (41) amino acid 1749, (42) amino acid 1796,

(43) amino acid 1802, (44) amino acid 1827, (45) amino acid 1861,

(46) amino acid 1896, (47) amino acid 1900, (48) amino acid 1904,

(49) amino acid 1905, (50) amino acid 1910, (51) amino acid 1937,

(52) amino acid 2019, (53) amino acid 2068, (54) amino acid 2111, (55) amino acid 2120, (56) amino acid 2171, (57) amino acid 2188,

(58) amino acid 2227, (59) amino acid 2277, and

(60) two or more combinations thereof.

In one embodiment, a FVIII protein useful for the invention comprises two

XTEN sequences, a first XTEN sequence inserted into a first XTEN insertion site and a second XTEN inserted into a second XTEN insertion site. Non-limiting examples of the first XTEN insertion site and the second XTEN insertion site are listed in Table 11.

TABLE 11. Exemplary Insertion Sites for Two XTENs

Insert i mi 1 Insert ion ~"

Insertion Site Domain Insertion Site Domain

1656 A3 1900 A3

1656 A3 1905 A3

1711 A3 40 Al

1711 A3 26 Al

1720 A3 26 Al

1720 A3 40 Al

1720 A3 18 Al

1725 A3 26 Al

1725 A3 40 Al

1725 A3 18 Al

1720 A3 403 A2

1720 A3 399 A2

1711 A3 403 A2

1720 A3 403 A2

1725 A3 403 A2

1725 A3 399 A2

1711 A3 403 A2

1900 A3 399 A2

1900 A3 403 A2

1905 A3 403 A2

1905 A3 399 A2

1910 A3 403 A2 The two XTENs inserted or linked to the FVIII protein can be identical or different. In some embodiments, a FVIII protein useful for the invention comprises two XTEN sequences inserted in the FVIII protein, a first XTEN sequence inserted immediately downstream of amino acid 745 corresponding to SEQ ID NO: 4, and a second XTEN sequence inserted immediately downstream of amino acid 2332 corresponding to SEQ ID NO: 4 (the C-terminus). In other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 18, 26, 40, 1656, or 1720 corresponding to SEQ ID NO: 4, and a second XTEN sequence inserted immediately downstream of amino acid 403 corresponding to SEQ ID NO: 4. In yet other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 18, 26, or 40 corresponding to SEQ ID NO: 4, and a second XTEN sequence inserted immediately downstream of amino acid 599 corresponding to SEQ ID NO: 4. In still other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO: 4, and a second XTEN sequence inserted immediately downstream of amino acid 18, 26, 40, 399, 403, 1725, 1720, 1900, 1905, or 2332 corresponding to SEQ ID NO: 4. In certain embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 1900 corresponding to SEQ ID NO: 4, and a second XTEN sequence inserted immediately downstream of amino acid 18, 26, or 40 corresponding to SEQ ID NO: 4. In some embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 18, 26, or 40 corresponding to SEQ ID NO: 4, and a second XTEN sequence inserted immediately downstream of amino acid 399 corresponding to SEQ ID NO: 4. In other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 1720 corresponding to SEQ ID NO: 4, and a second XTEN sequence inserted immediately

downstream of amino acid 18, 26, or 40 corresponding to SEQ ID NO: 4. In still other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 1720 corresponding to SEQ ID NO: 4, and a second XTEN sequence inserted immediately downstream of amino acid 18 corresponding to SEQ ID NO: 4. In a particular embodiment, the FVIII protein comprising two XTEN sequences, a first XTEN sequence inserted immediately downstream of amino acid 745 corresponding to SEQ ID NO: 4 and a second XTEN sequence inserted immediately downstream of amino acid 2332 corresponding to SEQ ID NO: 4, wherein the FVIII protein further has a deletion from amino acid 745 corresponding to SEQ ID NO: 4 to amino acid 1685 corresponding to SEQ ID NO: 4, a mutation or substitution at amino acid 1680 corresponding to SEQ ID NO: 4, e.g., Y1680F, a mutation or substitution at amino acid 1648 corresponding to SEQ ID NO: 4, e.g., R1648A, or at least two mutations or substitutions at amino acid 1648 corresponding to SEQ ID NO: 4, e.g., R1648A, and amino acid 1680 corresponding to SEQ ID NO: 4, e.g., Y1680F. In a specific embodiment, the FVIII protein comprises two XTEN sequences, a first XTEN inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO: 4 and a second XTEN sequence inserted immediately downstream of amino acid 2332 of SEQ ID NO: 4, wherein the FVIII protein further has a deletion from amino acid 745 to amino acid 1656 corresponding to SEQ ID NO: 4.

In certain embodiments, a FVIII protein comprises three XTEN sequences, a first XTEN sequence inserted into a first XTEN insertion site, a second XTEN sequence inserted into a second XTEN sequence, and a third XTEN sequence inserted into a third XTEN insertion site. The first, second, or third XTEN sequences can be identical or different. The first, second, and third insertion sites can be selected from the group of any one of the insertion sites disclosed herein. In some embodiments, the FVIII protein comprising three XTEN sequences can further comprise a mutation or substitution, e.g., amino acid 1648 corresponding to SEQ ID NO: 4, e.g., R1648A. For example, non-limiting examples of the first, second, and third XTEN insertion sites are listed in Table 12.

TABLE 12. Exemplary Insertion Sites for Three XTENs

In some embodiments, a FVIII protein comprises three XTEN sequences, a first XTEN sequence inserted immediately downstream of amino acid 26 corresponding to SEQ ID NO: 4, a second XTEN sequence inserted downstream of amino acid 403 corresponding to SEQ ID NO: 4, and a third XTEN sequence inserted downstream of amino acid 1656, 1720, or 1900 corresponding to SEQ ID NO: 4. In other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 26 corresponding to SEQ ID NO: 4, a second XTEN sequence is inserted downstream of amino acid 1656 corresponding to SEQ ID NO: 4, and a third XTEN sequence is inserted downstream of amino acid 1720 or 1900 corresponding to SEQ ID NO: 4. In yet other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 26 corresponding to SEQ ID NO: 4, a second XTEN sequence is inserted downstream of amino acid 1720 corresponding to SEQ ID NO: 4, and a third XTEN sequence is inserted downstream of amino acid 1900 corresponding to SEQ ID NO: 4. In still other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 403 corresponding to SEQ ID NO: 4, a second XTEN sequence is inserted downstream of amino acid 1656 corresponding to SEQ ID NO: 4, and a third XTEN sequence is inserted downstream of amino acid 1720 or 1900 corresponding to SEQ ID NO: 4. In other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 403 or 1656 corresponding to SEQ ID NO: 4, a second XTEN sequence is inserted downstream of amino acid 1720 corresponding to SEQ ID NO: 4, and a third XTEN sequence is inserted downstream of amino acid 1900 corresponding to SEQ ID NO: 4. In other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 18, 26, 40, 399, 403, 1711, 1720, 1725, 1900, 1905, or 1910 corresponding to SEQ ID NO: 4, a second XTEN sequence is inserted

downstream of amino acid 745 corresponding to SEQ ID NO: 4, and a third XTEN sequence is inserted downstream of amino acid 2332 corresponding to SEQ ID NO: 4.

In other embodiments, a FVIII protein in the invention comprises four

XTEN sequences, a first XTEN sequence inserted into a first insertion site, a second XTEN sequence inserted into a second insertion site, a third XTEN sequence inserted into a third insertion site, and a fourth XTEN sequence inserted into a fourth insertion site. The first, second, third, and fourth XTEN sequences can be identical, different, or combinations thereof. In some embodiments, the FVIII protein comprising four XTEN sequences can further comprise a mutation or substitution, e.g., amino acid 1648 corresponding to SEQ ID NO: 4, e.g., R1648A. Non-limiting examples of the first, second, third, and fourth XTEN insertion sites are listed in Table 13.

TABLE 13. Exemplary Insertion Sites for Four XTENs

0403 A2 1720 A3 745 B 2332 CT

0403 A2 1900 A3 745 B 2332 CT

0409 A2 1900 A3 745 B 2332 CT

0403 A2 1900 A3 745 B 2332 CT

0409 A2 1900 A3 745 B 2332 CT

0409 A2 1905 A3 745 B 2332 CT

0403 A2 1905 A3 745 B 2332 CT

0409 A2 1905 A3 745 B 2332 CT

0403 A2 1905 A3 745 B 2332 CT

0409 A2 1910 A3 745 B 2332 CT

0403 A2 1910 A3 745 B 2332 CT

1720 A3 1900 A3 745 B 2332 CT

1720 A3 1905 A3 745 B 2332 CT

1720 A3 1910 A3 745 B 2332 CT

0403 A2 1656 a3 1720 A3 2332 CT

0403 A2 1656 a3 1900 A3 2332 CT

0403 A2 1720 A3 1900 A3 2332 CT

1656 a3 1720 A3 1900 A3 2332 CT

0018 Al 0403 A2 1656 a3 2332 CT

0018 Al 0403 A2 1720 A3 2332 CT

0018 Al 0403 A2 1900 A3 2332 CT

0018 Al 1656 a3 1720 A3 2332 CT

0018 Al 1656 a3 1900 A3 2332 CT

0018 Al 1720 A3 1900 A3 2332 CT

0018 Al 0403 A2 0745 B 2332 CT

0018 Al 0745 B 1720 A3 2332 CT

0018 Al 0745 B 1900 A3 2332 CT

0403 A2 0745 B 1720 A3 2332 CT

0403 A2 0745 B 1900 A3 2332 CT

0745 B 1720 A3 1900 A3 2332 CT

0188 Al 1900 A3 0745 B 2332 CT

0599 1900 A3 0745 B 2332 CT

2068 1900 A3 0745 B 2332 CT

2171 1900 A3 0745 B 2332 CT

2227 1900 A3 0745 B 2332 CT

2277 1900 A3 0745 B 2332 CT

In some embodiments, a FVIII protein comprises five XTEN sequences, a first XTEN sequence inserted into a first insertion site, a second XTEN sequence inserted into a second insertion site, a third XTEN sequence inserted into a third XTEN insertion site, a fourth XTEN sequence inserted into a fourth XTEN insertion site, and a fifth XTEN sequence inserted into a fifth XTEN insertion site. The first, second, third, fourth, of fifth XTEN sequences can be identical, different, or combinations thereof. Non-limiting examples of the first, second, third, fourth, and fifth insertion sites are listed in Table 14.

TABLE 14. Exemplary Insertion Sites for Five XTENs

[0206] In certain embodiments, a FVIII protein comprises six XTEN sequences, a first XTEN sequence inserted into a first XTEN insertion site, a second XTEN sequence inserted into a second XTEN insertion site, a third XTEN sequence inserted into a third XTEN insertion site, a fourth XTEN sequence inserted into a fourth XTEN insertion site, a fifth XTEN sequence inserted into a fifth XTEN insertion site, and a sixth XTEN sequence inserted into a sixth XTEN insertion site. The first, second, third, fourth, fifth, or sixth XTEN sequences can be identical, different, or combinations thereof. Examples of the six XTEN insertion sites include, but are not limited to the insertion sites listed in Table 15.

TABLE 15. Exemplary XTEN Insertion Sites for Six XTENs

In a particular example, a first XTEN is inserted between amino acids 26 and 27 corresponding to SEQ ID NO: 4, and a second XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO: 4 (full-length mature FVIII). In another example, a first XTEN is inserted between amino acids 403 and 404 corresponding to SEQ ID NO: 4, and a second XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO: 4. In some examples, a first XTEN is inserted between amino acids 1656 and 1657 corresponding to SEQ ID NO: 4, and a second XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO: 4. In other examples, a first XTEN is inserted between amino acids 26 and 27 corresponding to SEQ ID NO: 4, a second XTEN is inserted between amino acids 1656 and 1657 corresponding to SEQ ID NO: 4, and a third XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO: 4. In yet other embodiments, a first XTEN is inserted between amino acids 403 and 404 corresponding to SEQ ID NO: 4, a second XTEN is inserted between amino acids 1656 and 1657 corresponding to SEQ ID NO: 4, and a third XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO: 4. In still other embodiments, a first XTEN is inserted between amino acids 403 and 404 corresponding to SEQ ID NO: 4, a second XTEN is inserted between amino acids 1656 and 1657 corresponding to SEQ ID NO: 4, and a third XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO: 4. In certain embodiments, a first XTEN is inserted between amino acids 26 and 27 corresponding to SEQ ID NO: 4, a second XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO: 4, and a third XTEN is inserted between amino acids 1900 and 1901 corresponding to SEQ ID NO: 4. In some embodiments, a first XTEN is inserted between amino acids 26 and 27 corresponding to SEQ ID NO: 4, a second XTEN is inserted between amino acids 1656 and 1657 corresponding to SEQ ID NO: 4, a third XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO: 4, and a fourth XTEN is inserted between 1900 and 1901 corresponding to SEQ ID NO: 4.

[0208] In a particular embodiment, an XTEN sequence is inserted between amino acids 745 and 746 of a full-length Factor VIII or the corresponding insertion site of the B-domain deleted Factor VIII.

[0209] In some embodiments, a chimeric protein of the invention comprises two polypeptide sequences, a first polypeptide sequence comprising an amino acid sequence at least about 80%, 90%, 95%, or 100% identical to a sequence selected from FVIII-161 (SEQ ID NO: 101), FVIII-169 (SEQ ID NO: 103), FVIII-170 (SEQ ID NO: 102), FVIII-173 (SEQ ID NO: 104); FVIII-195 (SEQ ID NO: 105); FVIII-196 (SEQ ID NO: 106), FVIII-199 (SEQ ID NO: 107), FVIII-201 (SEQ ID NO: 108); FVIII-203 (SEQ ID NO: 109), FVIII-204 (SEQ ID NO: 110), FVIII- 205 (SEQ ID NO: 111), FVIII-266 (SEQ ID NO: 112), FVIII-267 (SEQ ID NO: 113), FVIII-268 (SEQ ID NO: 114), FVIII-269 (SEQ ID NO: 115), FVIII-271 (SEQ ID NO: 116), or FVIII-272 (SEQ ID NO: 117) and a second polypeptide sequence comprising an amino acid sequence at least about 80%, 90%, 95%>, or 100% identical to a sequence selected from VWF031 (SEQ ID NO: 118), VWF034 (SEQ ID NO: 1 19), or VWF-036 (SEQ ID NO: 120).

D) Ig Constant Region or a portion thereof

[0210] The VWF fragment or the FVIII protein linked to an XTEN sequence in the present invention can further comprise an Ig constant region or a portion thereof. The Ig constant region or a portion thereof can improve pharmacokinetic or pharmacodynamic properties of the VWF fragment or the FVIII protein in combination with the XTEN sequence. In certain embodiments, the Ig constant region or a portion thereof extends a half-life of a molecule fused to the Ig constant region or a portion thereof.

[0211] An Ig constant region is comprised of domains denoted CH (constant heavy) domains (CHI, CH2, etc.). Depending on the isotype, (i.e. IgG, IgM, IgA, IgD, or IgE), the constant region can be comprised of three or four CH domains. Some isotypes (e.g. IgG) constant regions also contain a hinge region. See Janeway et al. 2001, Immunobiology, Garland Publishing, N.Y., N.Y.

[0212] An Ig constant region or a portion thereof for producing the chimeric protein of the present invention may be obtained from a number of different sources. In some embodiments, an Ig constant region or a portion thereof is derived from a human Ig. It is understood, however, that the Ig constant region or a portion thereof may be derived from an Ig of another mammalian species, including for example, a rodent (e.g. a mouse, rat, rabbit, guinea pig) or non- human primate (e.g. chimpanzee, macaque) species. Moreover, the Ig constant region or a portion thereof may be derived from any Ig class, including IgM, IgG, IgD, IgA, and IgE, and any Ig isotype, including IgGl, IgG2, IgG3, and IgG4. In one embodiment, the human isotype IgGl is used.

[0213] A variety of the Ig constant region gene sequences (e.g., human constant region gene sequences) are available in the form of publicly accessible deposits. Constant region domains sequence can be selected having a particular effector function (or lacking a particular effector function) or with a particular

modification to reduce immunogenicity. Many sequences of antibodies and antibody-encoding genes have been published and suitable Ig constant region sequences (e.g., hinge, CH2, and/or CH3 sequences, or portions thereof) can be derived from these sequences using art recognized techniques. The genetic material obtained using any of the foregoing methods may then be altered or synthesized to obtain polypeptides of the present invention. It will further be appreciated that the scope of this invention encompasses alleles, variants and mutations of constant region DNA sequences.

[0214] The sequences of the Ig constant region or a portion thereof can be cloned, e.g., using the polymerase chain reaction and primers which are selected to amplify the domain of interest. To clone a sequence of the Ig constant region or a portion thereof from an antibody, mRNA can be isolated from hybridoma, spleen, or lymph cells, reverse transcribed into DNA, and antibody genes amplified by PCR. PCR amplification methods are described in detail in U.S. Pat. Nos.

4,683,195; 4,683,202; 4,800,159; 4,965,188; and in, e.g., "PCR Protocols: A Guide to Methods and Applications" Innis et al. eds., Academic Press, San Diego, CA (1990); Ho et al. 1989. Gene 77:51 ; Horton et al. 1993. Methods Enzymol. 217:270). PCR may be initiated by consensus constant region primers or by more specific primers based on the published heavy and light chain DNA and amino acid sequences. As discussed above, PCR also may be used to isolate DNA clones encoding the antibody light and heavy chains. In this case the libraries may be screened by consensus primers or larger homologous probes, such as mouse constant region probes. Numerous primer sets suitable for amplification of antibody genes are known in the art (e.g., 5' primers based on the N-terminal sequence of purified antibodies (Benhar and Pastan. 1994. Protein Engineering 7: 1509); rapid amplification of cDNA ends (Ruberti, F. et al. 1994. J. Immunol. Methods 173 :33); antibody leader sequences (Larrick et al. 1989 Biochem. Biophys. Res. Commun. 160: 1250). The cloning of antibody sequences is further described in Newman et al, U.S. Pat. No. 5,658,570, filed January 25, 1995, which is incorporated by reference herein.

[0215] An Ig constant region used herein can include all domains and the hinge region or portions thereof. In one embodiment, the Ig constant region or a portion thereof comprises CH2 domain, CH3 domain, and a hinge region, i.e., an Fc region or an FcRn binding partner.

[0216] As used herein, the term "Fc region" is defined as the portion of a

polypeptide which corresponds to the Fc region of native Ig, i.e., as formed by the dimeric association of the respective Fc domains of its two heavy chains. A native Fc region forms a homodimer with another Fc region. In contrast, the term "genetically-fused Fc region" or "single-chain Fc region" (scFc region), as used herein, refers to a synthetic dimeric Fc region comprised of Fc domains genetically linked within a single polypeptide chain {i.e., encoded in a single contiguous genetic sequence).

[0217] In one embodiment, the "Fc region" refers to the portion of a single Ig heavy chain beginning in the hinge region just upstream of the papain cleavage site {i.e. residue 216 in IgG, taking the first residue of heavy chain constant region to be 114) and ending at the C-terminus of the antibody. Accordingly, a complete Fc domain comprises at least a hinge domain, a CH2 domain, and a CH3 domain.

[0218] The Fc region of an Ig constant region, depending on the Ig isotype can include the CH2, CH3, and CH4 domains, as well as the hinge region. Chimeric proteins comprising an Fc region of an Ig bestow several desirable properties on a chimeric protein including increased stability, increased serum half-life (see Capon et al, 1989, Nature 337:525) as well as binding to Fc receptors such as the neonatal Fc receptor (FcRn) (U.S. Pat. Nos. 6,086,875, 6,485,726, 6,030,613; WO 03/077834; US2003-0235536A1), which are incorporated herein by reference in their entireties.

[0219] An Ig constant region or a portion thereof can be an FcRn binding partner.

FcRn is active in adult epithelial tissues and expressed in the lumen of the intestines, pulmonary airways, nasal surfaces, vaginal surfaces, colon and rectal surfaces (U.S. Pat. No. 6,485,726). An FcRn binding partner is a portion of an Ig that binds to FcRn. [0220] The FcRn receptor has been isolated from several mammalian species including humans. The sequences of the human FcRn, monkey FcRn, rat FcRn, and mouse FcRn are known (Story et al. 1994, J. Exp. Med. 180:2377). The FcRn receptor binds IgG (but not other Ig classes such as IgA, IgM, IgD, and IgE) at relatively low pH, actively transports the IgG transcellularly in a luminal to serosal direction, and then releases the IgG at relatively higher pH found in the interstitial fluids. It is expressed in adult epithelial tissue (U.S. Pat. Nos.

6,485,726, 6,030,613, 6,086,875; WO 03/077834; US2003-0235536A1) including lung and intestinal epithelium (Israel et al. 1997, Immunology 92:69) renal proximal tubular epithelium (Kobayashi et al. 2002, Am. J. Physiol. Renal Physiol. 282:F358) as well as nasal epithelium, vaginal surfaces, and biliary tree surfaces.

[0221] FcRn binding partners useful in the present invention encompass

molecules that can be specifically bound by the FcRn receptor including whole IgG, the Fc fragment of IgG, and other fragments that include the complete binding region of the FcRn receptor. The region of the Fc portion of IgG that binds to the FcRn receptor has been described based on X-ray crystallography (Burmeister et al. 1994, Nature 372:379). The major contact area of the Fc with the FcRn is near the junction of the CH2 and CH3 domains. Fc-FcRn contacts are all within a single Ig heavy chain. The FcRn binding partners include whole IgG, the Fc fragment of IgG, and other fragments of IgG that include the complete binding region of FcRn. The major contact sites include amino acid residues 248, 250-257, 272, 285, 288, 290-291, 308-311, and 314 of the CH2 domain and amino acid residues 385-387, 428, and 433-436 of the CH3 domain. References made to amino acid numbering of Igs or Ig fragments, or regions, are all based on Kabat et al. 1991, Sequences of Proteins of Immunological Interest, U.S. Department of Public Health, Bethesda, Md.

[0222] Fc regions or FcRn binding partners bound to FcRn can be effectively shuttled across epithelial barriers by FcRn, thus providing a non-invasive means to systemically administer a desired therapeutic molecule. Additionally, fusion proteins comprising an Fc region or an FcRn binding partner are endocytosed by cells expressing the FcRn. But instead of being marked for degradation, these fusion proteins are recycled out into circulation again, thus increasing the in vivo half-life of these proteins. In certain embodiments, the portions of Ig constant regions are an Fc region or an FcRn binding partner that typically associates, via disulfide bonds and other non-specific interactions, with another Fc region or another FcRn binding partner to form dimers and higher order multimers.

[0223] Two FcRn receptors can bind a single Fc molecule. Crystallo graphic data suggest that each FcRn molecule binds a single polypeptide of the Fc homodimer. In one embodiment, linking the FcRn binding partner, e.g., an Fc fragment of an IgG, to a biologically active molecule provides a means of delivering the biologically active molecule orally, buccally, sublingually, rectally, vaginally, as an aerosol administered nasally or via a pulmonary route, or via an ocular route. In another embodiment, the chimeric protein can be administered invasively, e.g., subcutaneously, intravenously.

[0224] An FcRn binding partner region is a molecule or a portion thereof that can be specifically bound by the FcRn receptor with consequent active transport by the FcRn receptor of the Fc region. Specifically bound refers to two molecules forming a complex that is relatively stable under physiologic conditions. Specific binding is characterized by a high affinity and a low to moderate capacity as distinguished from nonspecific binding which usually has a low affinity with a moderate to high capacity. Typically, binding is considered specific when the affinity constant KA is higher than 10⁶ M^"1, or higher than 10⁸ M^"1. If necessary, non-specific binding can be reduced without substantially affecting specific binding by varying the binding conditions. The appropriate binding conditions such as concentration of the molecules, ionic strength of the solution, temperature, time allowed for binding, concentration of a blocking agent {e.g. serum albumin, milk casein), etc., may be optimized by a skilled artisan using routine techniques.

[0225] In certain embodiments, a chimeric protein of the invention comprises one or more truncated Fc regions that are nonetheless sufficient to confer Fc receptor (FcR) binding properties to the Fc region. For example, the portion of an Fc region that binds to FcRn {i.e., the FcRn binding portion) comprises from about amino acids 282-438 of IgGl, EU numbering (with the primary contact sites being amino acids 248, 250-257, 272, 285, 288, 290-291, 308-311, and 314 of the CH2 domain and amino acid residues 385-387, 428, and 433-436 of the CH3 domain. Thus, an Fc region of the invention may comprise or consist of an FcRn binding portion. FcRn binding portions may be derived from heavy chains of any isotype, including IgGl, IgG2, IgG3 and IgG4. In one embodiment, an FcRn binding portion from an antibody of the human isotype IgGl is used. In another embodiment, an FcRn binding portion from an antibody of the human isotype IgG4 is used.

[0226] In another embodiment, the "Fc region" includes an amino acid sequence of an Fc domain or derived from an Fc domain. In certain embodiments, an Fc region comprises at least one of: a hinge (e.g., upper, middle, and/or lower hinge region) domain (about amino acids 216-230 of an antibody Fc region according to EU numbering), a CH2 domain (about amino acids 231-340 of an antibody Fc region according to EU numbering), a CH3 domain (about amino acids 341-438 of an antibody Fc region according to EU numbering), a CH4 domain, or a variant, portion, or fragment thereof. In other embodiments, an Fc region comprises a complete Fc domain (i.e., a hinge domain, a CH2 domain, and a CH3 domain). In some embodiments, an Fc region comprises, consists essentially of, or consists of a hinge domain (or a portion thereof) fused to a CH3 domain (or a portion thereof), a hinge domain (or a portion thereof) fused to a CH2 domain (or a portion thereof), a CH2 domain (or a portion thereof) fused to a CH3 domain (or a portion thereof), a CH2 domain (or a portion thereof) fused to both a hinge domain (or a portion thereof) and a CH3 domain (or a portion thereof). In still other embodiments, an Fc region lacks at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). In a particular embodiment, an Fc region comprises or consists of amino acids corresponding to EU numbers 221 to 447.

[0227] The Fc regions denoted as F, Fl, or F2 herein may be obtained from a number of different sources. In one embodiment, an Fc region of the polypeptide is derived from a human Ig. It is understood, however, that an Fc region may be derived from an Ig of another mammalian species, including for example, a rodent (e.g. a mouse, rat, rabbit, or guinea pig) or non-human primate (e.g. chimpanzee, macaque) species. Moreover, the polypeptide of the Fc domains or portions thereof may be derived from any Ig class, including IgM, IgG, IgD, IgA and IgE, and any Ig isotype, including IgGl, IgG2, IgG3 and IgG4. In another embodiment, the human isotype IgGl is used. [0228] In certain embodiments, the Fc variant confers a change in at least one effector function imparted by an Fc region comprising said wild-type Fc domain (e.g., an improvement or reduction in the ability of the Fc region to bind to Fc receptors (e.g. FcyRI, FcyRII, or FcyRIII) or complement proteins (e.g. C lq), or to trigger antibody-dependent cytotoxicity (ADCC), phagocytosis, or

complement-dependent cytotoxicity (CDCC)). In other embodiments, the Fc variant provides an engineered cysteine residue.

[0229] The Fc regions of the invention may employ art-recognized Fc variants which are known to impart a change (e.g., an enhancement or reduction) in effector function and/or FcR or FcRn binding. Specifically, a binding molecule of the invention may include, for example, a change (e.g., a substitution) at one or more of the amino acid positions disclosed in International PCT Publications WO88/07089A1 , W096/14339A1 , WO98/05787A1 , W098/23289A1 ,

W099/51642A1 , W099/58572A1 , WO00/09560A2, WO00/32767A1 ,

WO00/42072A2, WO02/44215A2, WO02/060919A2, WO03/074569A2,

WO04/016750A2, WO04/029207A2, WO04/035752A2, WO04/063351A2, WO04/074455A2, WO04/099249A2, WO05/040217A2, WO04/044859,

WO05/070963A1 , WO05/077981A2, WO05/092925A2, WO05/123780A2, WO06/019447A1 , WO06/047350A2, and WO06/085967A2; US Patent

Publication Nos. US2007/0231329, US2007/0231329, US2007/0237765,

US2007/0237766, US2007/0237767, US2007/0243188, US20070248603, US20070286859, US20080057056 ; or US Patents 5,648,260; 5,739,277;

5,834,250; 5,869,046; 6,096,871 ; 6, 121 ,022; 6,194,551 ; 6,242,195; 6,277,375; 6,528,624; 6,538,124; 6,737,056; 6,821 ,505; 6,998,253; 7,083,784; 7,404,956, and 7,317,091 , each of which is incorporated by reference herein. In one embodiment, the specific change (e.g., the specific substitution of one or more amino acids disclosed in the art) may be made at one or more of the disclosed amino acid positions. In another embodiment, a different change at one or more of the disclosed amino acid positions (e.g., the different substitution of one or more amino acid position disclosed in the art) may be made.

[0230] The Fc region or FcRn binding partner of IgG can be modified according to well recognized procedures such as site directed mutagenesis and the like to yield modified IgG or Fc fragments or portions thereof that will be bound by FcRn. Such modifications include modifications remote from the FcRn contact sites as well as modifications within the contact sites that preserve or even enhance binding to the FcRn. For example, the following single amino acid residues in human IgGl Fc (Fc γΐ) can be substituted without significant loss of Fc binding affinity for FcRn: P238A, S239A, K246A, K248A, D249A, M252A, T256A, E258A, T260A, D265A, S267A, H268A, E269A, D270A, E272A, L274A, N276A, Y278A, D280A, V282A, E283A, H285A, N286A, T289A, K290A, R292A, E293A, E294A, Q295A, Y296F, N297A, S298A, Y300F, R301A, V303A, V305A, T307A, L309A, Q311A, D312A, N315A, K317A, E318A, K320A, K322A, S324A, K326A, A327Q, P329A, A330Q, P331A, E333A, K334A, T335A, S337A, K338A, K340A, Q342A, R344A, E345A, Q347A, R355A, E356A, M358A, T359A, K360A, N361A, Q362A, Y373A, S375A, D376A, A378Q, E380A, E382A, S383A, N384A, Q386A, E388A, N389A, N390A, Y391F, K392A, L398A, S400A, D401A, D413A, K414A, R416A, Q418A, Q419A, N421A, V422A, S424A, E430A, N434A, T437A, Q438A, K439A, S440A, S444A, and K447A, where for example P238A represents wild type proline substituted by alanine at position number 238. As an example, a specific embodiment incorporates the N297A mutation, removing a highly conserved N-glycosylation site. In addition to alanine other amino acids may be substituted for the wild type amino acids at the positions specified above. Mutations may be introduced singly into Fc giving rise to more than one hundred Fc regions distinct from the native Fc. Additionally, combinations of two, three, or more of these individual mutations may be introduced together, giving rise to hundreds more Fc regions. Moreover, one of the Fc region of a construct of the invention may be mutated and the other Fc region of the construct not mutated at all, or they both may be mutated but with different mutations.

Certain of the above mutations may confer new functionality upon the Fc region or FcRn binding partner. For example, one embodiment incorporates N297A, removing a highly conserved N-glycosylation site. The effect of this mutation is to reduce immunogenicity, thereby enhancing circulating half-life of the Fc region, and to render the Fc region incapable of binding to FcyRI, FcyRIIA, FcyRIIB, and FcyRIIIA, without compromising affinity for FcRn (Routledge et al. 1995, Transplantation 60:847; Friend et al. 1999, Transplantation 68: 1632; Shields et al. 1995, J. Biol. Chem. 276:6591). As a further example of new functionality arising from mutations described above affinity for FcRn may be increased beyond that of wild type in some instances. This increased affinity may reflect an increased "on" rate, a decreased "off rate or both an increased "on" rate and a decreased "off rate. Examples of mutations believed to impart an increased affinity for FcRn include, but not limited to, T256A, T307A, E380A, and N434A (Shields et al. 2001, J. Biol. Chem. 276:6591).

[0232] Additionally, at least three human Fc gamma receptors appear to recognize a binding site on IgG within the lower hinge region, generally amino acids 234- 237. Therefore, another example of new functionality and potential decreased immunogenicity may arise from mutations of this region, as for example by replacing amino acids 233-236 of human IgGl "ELLG" to the corresponding sequence from IgG2 "PVA" (with one amino acid deletion). It has been shown that FcyRI, FcyRII, and FcyRIII, which mediate various effector functions will not bind to IgGl when such mutations have been introduced. Ward and Ghetie 1995, Therapeutic Immunology 2:77 and Armour et al. 1999, Eur. J. Immunol. 29:2613.

[0233] In one embodiment, the Ig constant region or a portion thereof, e.g, an Fc region, is a polypeptide including the sequence PKNSSMISNTP (SEQ ID NO: 52) and optionally further including a sequence selected from HQSLGTQ (SEQ ID NO: 53), HQNLSDGK (SEQ ID NO: 54), HQNISDGK (SEQ ID NO: 55), or VISSHLGQ (SEQ ID NO: 56) (U.S. Pat. No. 5,739,277).

[0234] In another embodiment, the immunoglobulin constant region or a portion thereof comprises an amino acid sequence in the hinge region or a portion thereof that forms one or more disulfide bonds with another immunoglobulin constant region or a portion thereof. The disulfide bond by the immunoglobulin constant region or a portion thereof places the first polypeptide comprising FVIII and the second polypeptide comprising the VWF fragment together so that endogenous VWF does not replace the VWF fragment and does not bind to the FVIII.

Therefore, the disulfide bond between the first immunoglobulin constant region or a portion thereof and a second immunoglobulin constant region or a portion thereof prevents interaction between endogenous VWF and the FVIII protein. This inhibition of interaction between the VWF and the FVIII protein allows the half-life of the FVIII protein to go beyond the two fold limit. The hinge region or a portion thereof can further be linked to one or more domains of CHI, CH2, CH3, a fragment thereof, and any combinations thereof. In a particular embodiment, the immunoglobulin constant region or a portion thereof is a hinge region and CH2.

[0235] In certain embodiments, the Ig constant region or a portion thereof is hemi- glycosylated. For example, the chimeric protein comprising two Fc regions or FcRn binding partners may contain a first, glycosylated, Fc region (e.g., a glycosylated CH2 region) or FcRn binding partner and a second, aglycosylated, Fc region (e.g., an aglycosylated CH2 region) or FcRn binding partner. In one embodiment, a linker may be interposed between the glycosylated and

aglycosylated Fc regions. In another embodiment, the Fc region or FcRn binding partner is fully glycosylated, i.e., all of the Fc regions are glycosylated. In other embodiments, the Fc region may be aglycosylated, i.e., none of the Fc moieties are glycosylated.

[0236] In certain embodiments, a chimeric protein of the invention comprises an amino acid substitution to an Ig constant region or a portion thereof (e.g., Fc variants), which alters the antigen-independent effector functions of the Ig constant region, in particular the circulating half-life of the protein.

[0237] Such proteins exhibit either increased or decreased binding to FcRn when compared to proteins lacking these substitutions and, therefore, have an increased or decreased half-life in serum, respectively. Fc variants with improved affinity for FcRn are anticipated to have longer serum half-lives, and such molecules have useful applications in methods of treating mammals where long half-life of the administered polypeptide is desired, e.g., to treat a chronic disease or disorder (see, e.g., US Patents 7,348,004, 7,404,956, and 7,862,820). In contrast, Fc variants with decreased FcRn binding affinity are expected to have shorter half- lives, and such molecules are also useful, for example, for administration to a mammal where a shortened circulation time may be advantageous, e.g. for in vivo diagnostic imaging or in situations where the starting polypeptide has toxic side effects when present in the circulation for prolonged periods. Fc variants with decreased FcRn binding affinity are also less likely to cross the placenta and, thus, are also useful in the treatment of diseases or disorders in pregnant women. In addition, other applications in which reduced FcRn binding affinity may be desired include those applications in which localization the brain, kidney, and/or liver is desired. In one exemplary embodiment, the chimeric protein of the invention exhibit reduced transport across the epithelium of kidney glomeruli from the vasculature. In another embodiment, the chimeric protein of the invention exhibit reduced transport across the blood brain barrier (BBB) from the brain, into the vascular space. In one embodiment, a protein with altered FcRn binding comprises at least one Fc region or FcRn binding partner (e.g, one or two Fc regions or FcRn binding partners) having one or more amino acid substitutions within the "FcRn binding loop" of an Ig constant region. The FcRn binding loop is comprised of amino acid residues 280-299 (according to EU numbering) of a wild-type, full-length, Fc region. In other embodiments, an Ig constant region or a portion thereof in a chimeric protein of the invention having altered FcRn binding affinity comprises at least one Fc region or FcRn binding partner having one or more amino acid substitutions within the 15 A FcRn "contact zone." As used herein, the term 15 A FcRn "contact zone" includes residues at the following positions of a wild-type, full-length Fc moiety: 243-261 , 275-280, 282-293, 302- 319, 336- 348, 367, 369, 372-389, 391 , 393, 408, 424, 425-440 (EU numbering). In other embodiments, a Ig constant region or a portion thereof of the invention having altered FcRn binding affinity comprises at least one Fc region or FcRn binding partner having one or more amino acid substitutions at an amino acid position corresponding to any one of the following EU positions: 256, 277-281 , 283-288, 303-309, 313, 338, 342, 376, 381 , 384, 385, 387, 434 (e.g., N434A or N434K), and 438. Exemplary amino acid substitutions which altered FcRn binding activity are disclosed in International PCT Publication No. WO05/047327 which is incorporated by reference herein.

An Fc region or FcRn binding partner used in the invention may also comprise an art recognized amino acid substitution which alters the glycosylation of the chimeric protein. For example, the Fc region or FcRn binding partner of the chimeric protein linked to a VWF fragment or a FVIII protein may comprise an Fc region having a mutation leading to reduced glycosylation (e.g., N- or O-linked glycosylation) or may comprise an altered glycoform of the wild-type Fc moiety (e.g., a low fucose or fucose-free glycan). [0239] In one embodiment, an unprocessed chimeric protein of the invention may comprise a genetically fused Fc region (i.e., scFc region) having two or more of its constituent Ig constant region or a portion thereof independently selected from the Ig constant region or a portion thereof described herein. In one embodiment, the Fc regions of a dimeric Fc region are the same. In another embodiment, at least two of the Fc regions are different. For example, the Fc regions or FcRn binding partners of the proteins of the invention comprise the same number of amino acid residues or they may differ in length by one or more amino acid residues (e.g., by about 5 amino acid residues (e.g., 1, 2, 3, 4, or 5 amino acid residues), about 10 residues, about 15 residues, about 20 residues, about 30 residues, about 40 residues, or about 50 residues). In yet other embodiments, the Fc regions or FcRn binding partners of the protein of the invention may differ in sequence at one or more amino acid positions. For example, at least two of the Fc regions or FcRn binding partners may differ at about 5 amino acid positions (e.g., 1, 2, 3, 4, or 5 amino acid positions), about 10 positions, about 15 positions, about 20 positions, about 30 positions, about 40 positions, or about 50 positions).

E) Linkers

[0240] The chimeric protein of the present invention further comprises one or more linkers. One type of the linkers is a cleavable linker, which can be cleaved by various proteases when administered to a subject in vivo, e.g., at a site of coagulation. In one embodiment, the cleavable linker allows cleavage of moiety, e.g., a VWF fragment, from the chimeric protein at the site of the coagulation cascade, thus allowing activated FVIII (FVIIIa) to have its FVIIIa activity.

Another type of the linkers is a processable linker, which contains an intracellular cleavage site and thus can be cleaved by an intracellular processing enzyme in a host cell, allowing convenient expression of a polypeptide and formation of a chimeric protein.

[0241] One or more linkers can be present between any two proteins in the

chimeric protein. In one embodiment, a chimeric protein comprises (i) a VWF fragment, (ii) an XTEN sequence, and (iii) a FVIII protein, wherein the VWF fragment is linked to the XTEN sequence by a linker, e.g., a cleavable linker, and the XTEN sequence is further linked to the FVIII protein (i.e., V-L-X-FVIII). In another embodiment, a chimeric protein comprises (i) a VWF fragment, (ii) an XTEN sequence, and (iii) a FVIII protein, wherein the VWF fragment is linked to the XTEN sequence, and the XTEN sequence is linked to the FVIII protein by a linker, e.g., a cleavable linker {i.e., V-X-L-FVIII).

[0242] In certain embodiments, a chimeric protein comprises (i) a VWF fragment,

(ii) an XTEN sequence, (iii) a first Ig constant region or a portion thereof {e.g., a first Fc region), (iv) a FVIII protein, and (v) a second Ig constant region or a portion thereof {e.g., a second Fc region), wherein the VWF fragment is linked to the XTEN sequence by an optional linker, e.g., a cleavable linker. The XTEN sequence can be further linked to the first Ig constant region or a portion thereof by a linker, e.g., a cleavable linker. The FVIII protein (with or without an XTEN sequence) can also be linked to the second Ig constant region or a portion thereof by an optional linker, e.g. a cleavable linker. In certain embodiments, the chimeric protein further comprises one or more linkers, e.g., processable linkers, between the first Ig constant region or a portion thereof {e.g., first Fc region) and the second Ig constant region or a portion thereof {e.g., second Fc region), between the VWF fragment and the second Ig constant region or a portion thereof, or between the FVIII protein and the first Ig constant region or a portion thereof {e.g., first Fc region).

[0243] In some embodiments, the present invention includes a chimeric protein comprising (i) a FVIII protein, (ii) an XTEN sequence, (iii) a first Ig constant region or a portion thereof, and (iv) a second Ig constant region or a portion thereof, wherein the first Ig constant region or a portion thereof and the second Ig constant region or a portion thereof are linked by a processable linker.

[0244] The linker useful in the present invention can comprise any organic

molecule. In one embodiment, the linker comprises a polymer, e.g., polyethylene glycol (PEG) or hydroxyethyl starch (HES). In another embodiment, the linker comprises an amino acids sequence. The linker can comprise at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400 ,500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 amino acids. The linker can comprise 1-5 amino acids, 1-10 amino acids, 1-20 amino acids, 10-50 amino acids, 50-100 amino acids, 100-200 amino acids, 200-300 amino acids, 300-400 amino acids, 400-500 amino acids, 500-600 amino acids, 600-700 amino acids, 700-800 amino acids, 800-900 amino acids, or 900-1000 amino acids. In one embodiment, the linker comprises an XTEN sequence.

Additional examples of XTEN can be used according to the present invention and are disclosed in US Patent Publication Nos. 2010/0239554 Al, 2010/0323956 Al, 2011/0046060 Al, 2011/0046061 Al, 2011/0077199 Al, or 2011/0172146 Al, or International Patent Publication Nos. WO 2010091122 Al, WO 2010144502 A2, WO 2010144508 Al, WO 2011028228 Al, WO 2011028229 Al, or WO

2011028344 A2. In another embodiment, the linker is a PAS sequence.

[0245] The linker useful in the present invention can comprise any organic

molecule. In one embodiment, the linker is a polymer, e.g., polyethylene glycol (PEG) or hydroxyethyl starch (HES). In another embodiment, the linker is an amino acid sequence. The linker can comprise at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 amino acids. The linker can comprise 1-5 amino acids, 1-10 amino acids, 1-20 amino acids, 10-50 amino acids, 50-100 amino acids, 100-200 amino acids, 200-300 amino acids, 300-400 amino acids, 400-500 amino acids, 500-600 amino acids, 600-700 amino acids, 700-800 amino acids, 800-900 amino acids, or 900-1000 amino acids.

[0246] Examples of linkers are well known in the art. In one embodiment, the linker comprises the sequence G_n. The linker can comprise the sequence (GA)_n. The linker can comprise the sequence (GGS)_n. In other embodiments, the linker comprises (GGGS)_n (SEQ ID NO: 57). In still other embodiments, the linker comprises the sequence (GGS)_n(GGGGS)_n (SEQ ID NO: 58). In these instances, n may be an integer from 1-100. In other instances, n may be an integer from 1- 20, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

Examples of linkers include, but are not limited to, GGG, SGGSGGS (SEQ ID NO: 59), GGSGGSGGSGGSGGG (SEQ ID NO: 60), GGSGGSGGGGSGGGGS (SEQ ID NO: 61), GGSGGSGGSGGSGGSGGS (SEQ ID NO: 62), or

GGGGSGGGGSGGGGS (SEQ ID NO: 63). The linker does not eliminate or diminish the VWF fragment activity or the clotting activity of Factor VIII.

Optionally, the linker enhances the VWF fragment activity or the clotting activity of Factor VIII protein, e.g., by further diminishing the effects of steric hindrance and making the VWF fragment or Factor VIII portion more accessible to its target binding site. [0247] In one embodiment, the linker useful for the chimeric protein is 15-25 amino acids long. In another embodiment, the linker useful for the chimeric protein is 15-20 amino acids long. In some embodiments, the linker for the chimeric protein is 10-25 amino acids long. In other embodiments, the linker for the chimeric protein is 15 amino acids long. In still other embodiments, the linker for the chimeric protein is (GGGGS)_n (SEQ ID NO: 64) where G represents glycine, S represents serine and n is an integer from 1-20.

F) Cleavage Sites

[0248] The linker may also incorporate a moiety capable of being cleaved either chemically (e.g., hydrolysis of an ester bond), enzymatically (i.e., incorporation of a protease cleavage sequence), or photolytically (e.g., a chromophore such as 3- amino-3-(2-nitrophenyl) proprionic acid (ANP)) in order to release one molecule from another.

[0249] In one embodiment, the linker is a cleavable linker. The cleavable linkers can comprise one or more cleavage sites at the N-terminus or C-terminus or both. In another embodiment, the cleavable linker consists essentially of or consists of one or more cleavable sites. In other embodiments, the cleavable linker comprises heterologous amino acid linker sequences described herein or polymers and one or more cleavable sites.

[0250] In certain embodiments, a cleavable linker comprises one or more cleavage sites that can be cleaved in a host cell (i.e., intracellular processing sites). Non limiting examples of the cleavage site include RR R (SEQ ID NO: 9), R RRKR (SEQ ID NO: 10), and RRRRS (SEQ ID NO: 11).

[0251] In other embodiments, a cleavable linker comprises one or more cleavage sites that are cleaved by a protease after a chimeric protein comprising the cleavable linker is administered to a subject. In one embodiment, the cleavage site is cleaved by a protease selected from the group consisting of factor XIa, factor Xlla, kallikrein, factor Vila, factor IXa, factor Xa, factor Ila (thrombin), Elastase- 2, MMP-12, MMP-13, MMP-17, and MMP-20. In another embodiment, the cleavage site is selected from the group consisting of a FXIa cleavage site (e.g., KLTRjAET (SEQ ID NO: 65)), a FXIa cleavage site (e.g, DFTRjVVG (SEQ ID NO: 66)), a FXIIa cleavage site (e.g., TMTRjIVGG (SEQ ID NO: 67)), a

Kallikrein cleavage site (e.g., SPFRjSTGG (SEQ ID NO: 68)), a FVIIa cleavage site (e.g., LQVR4IVGG (SEQ ID NO: 69)), a FIXa cleavage site (e.g.,

PLGR4IVGG (SEQ ID NO: 70)), a FXa cleavage site (e.g., IEGRjTVGG (SEQ ID NO: 71)), a Flla (thrombin) cleavage site (e.g, LTPRjSLLV (SEQ ID NO: 72)), a Elastase-2 cleavage site (e.g, LGPV jSGVP (SEQ ID NO: 73)), a

Granzyme-B cleavage (e.g, VAGD jSLEE (SEQ ID NO: 74)), a MMP-12 cleavage site (e.g., GPAGjLGGA (SEQ ID NO: 75)), a MMP-13 cleavage site (e.g., GPAGjLRGA (SEQ ID NO: 76)), a MMP-17 cleavage site (e.g.,

APLGjLRLR (SEQ ID NO: 77)), a MMP-20 cleavage site (e.g., PALP jLVAQ (SEQ ID NO: 78)), a TEV cleavage site (e.g., ENLYFQjG (SEQ ID NO: 79)), a Enterokinase cleavage site (e.g., DDDKjIVGG (SEQ ID NO: 80)), a Protease 3C (PRESCISSION™) cleavage site (e.g., LEVLFQjGP (SEQ ID NO: 81)), and a Sortase A cleavage site (e.g., LPKTjGSES) (SEQ ID NO: 82). In certain embodiments, the FXIa cleavage sites include, but are not limited to, e.g., TQSFNDFTR (SEQ ID NO: 83) and SVSQTSKLTR (SEQ ID NO: 84). Non- limiting exemplary thrombin cleavage sites include, e.g., DFLAEGGGVR (SEQ ID NO: 85), TTKIKPR (SEQ ID NO: 86), or LVPRG (SEQ ID NO: 87), and a sequence comprising, consisting essentially of, or consisting of ALRPR (SEQ ID NO: 17) (e.g., ALRPRVVGGA (SEQ ID NO: 88)).

[0252] In a specific embodiment, the cleavage site is

TLDPRSFLLRNPNDKYEPFWEDEEK (SEQ ID NO: 8).

POLYNUCLEOTIDES, VECTORS, AND HOST CELLS

[0253] Also provided in the invention is a polynucleotide encoding (a) a VWF fragment linked to an XTEN sequence and a FVIII protein, (b) a FVIII protein linked to an XTEN sequence and Fc, or (c) a FVIII protein linked to an XTEN sequence and a VWF fragment described herein. When a chimeric protein is a single polypeptide chain (e.g., F2-L2-X-V-L1-F1 -FVIII, wherein FVIII comprises a FVIII protein, Fl comprises a first Ig constant region or a portion thereof, e.g., a first Fc region, LI comprises a first linker, V comprises a VWF fragment, X comprises an XTEN sequence, L2 comprises a second linker, and F2 comprises a second Ig constant region or a portion thereof, e.g., a second Fc region), the invention is drawn to a single polynucleotide chain encoding the single polypeptide chain. When the chimeric protein comprises a first and a second polypeptide chains (F2-L2-X-V:FVIII-F1), the first polypeptide chain comprising a VWF fragment linked to a XTEN sequence, which is further linked to a first Ig constant region or a portion thereof (e.g. , a first Fc region) by a cleavable linker (e.g., F2-L2-X-V) and the second polypeptide chain comprising a FVIII protein and a second Ig constant region or a portion thereof (e.g., a second Fc region) (e.g, FVIII-F1), wherein the first polypeptide chain and the second polypeptide chain are associated with each other, a polynucleotide can comprise the first nucleotide sequence and the second nucleotide sequence. In one embodiment, the first polypeptide chain and the second polypeptide chain can be encoded by a single polynucleotide chain. In another embodiment, the first polypeptide chain and the second polypeptide chain are encoded by two different polynucleotides, i.e., a first nucleotide sequence and a second nucleotide sequence. In another embodiment, the first nucleotide sequence and the second nucleotide sequence are on two different polynucleotides (e.g., different vectors). In certain embodiments, the present invention is directed to a set of polynucleotides comprising a first nucleotide chain and a second nucleotide chain, wherein the first nucleotide chain encodes the VWF fragment of the chimeric protein and the second nucleotide chain encodes the FVIII protein. In some embodiments, a chimeric protein comprising two polypeptide chains or three polypeptide chains can be encoded by a single polynucleotide chain, and then processed into two or three (or more) polypeptide chains. In yet other embodiments, a chimeric protein comprising these polypeptide chains can be encoded by two or three polynucleotide chains.

In other embodiments, the set of the polynucleotides further comprises an additional nucleotide chain (e.g. , a second nucleotide chain when the chimeric polypeptide is encoded by a single polynucleotide chain or a third nucleotide chain when the chimeric protein is encoded by two polynucleotide chains) which encodes a protein convertase. The protein convertase can be selected from the group consisting of proprotein convertase subtilisin/kexin type 5 (PCSK5 or PC5), proprotein convertase subtilisin/kexin type 7 (PCSK7 or PC5), a yeast Kex 2, proprotein convertase subtilisin/kexin type 3 (PACE or PCSK3), and two or more combinations thereof. In some embodiments, the protein convertase is PACE, PC5, or PC7. In a specific embodiment, the protein convertase is PC5 or PC7. See International Application no. PCT/US2011/043568. [0255] As used herein, an expression vector refers to any nucleic acid construct which contains the necessary elements for the transcription and translation of an inserted coding sequence, or in the case of an RNA viral vector, the necessary elements for replication and translation, when introduced into an appropriate host cell. Expression vectors can include plasmids, phagemids, viruses, and derivatives thereof.

[0256] Expression vectors of the invention will include polynucleotides encoding the chimeric protein described herein. In one embodiment, one or more of the coding sequences for the VWF fragment and XTEN, the FVIII protein and XTEN, or both are operably linked to an expression control sequence. As used herein, two nucleic acid sequences are operably linked when they are covalently linked in such a way as to permit each component nucleic acid sequence to retain its functionality. A coding sequence and a gene expression control sequence are said to be operably linked when they are covalently linked in such a way as to place the expression or transcription and/or translation of the coding sequence under the influence or control of the gene expression control sequence. Two DNA sequences are said to be operably linked if induction of a promoter in the 5' gene expression sequence results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a gene expression sequence would be operably linked to a coding nucleic acid sequence if the gene expression sequence were capable of effecting transcription of that coding nucleic acid sequence such that the resulting transcript is translated into the desired protein or polypeptide.

[0257] A gene expression control sequence as used herein is any regulatory

nucleotide sequence, such as a promoter sequence or promoter-enhancer combination, which facilitates the efficient transcription and translation of the coding nucleic acid to which it is operably linked. The gene expression control sequence may, for example, be a mammalian or viral promoter, such as a constitutive or inducible promoter. Constitutive mammalian promoters include, but are not limited to, the promoters for the following genes: hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, beta- actin promoter, and other constitutive promoters. Exemplary viral promoters which function constitutively in eukaryotic cells include, for example, promoters from the cytomegalovirus (CMV), simian virus (e.g., SV40), papilloma virus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, the long terminal repeats (LTR) of Moloney leukemia virus, and other retroviruses, and the thymidine kinase promoter of herpes simplex virus. Other constitutive promoters are known to those of ordinary skill in the art. The promoters useful as gene expression sequences of the invention also include inducible promoters. Inducible promoters are expressed in the presence of an inducing agent. For example, the metallothionein promoter is induced to promote transcription and translation in the presence of certain metal ions. Other inducible promoters are known to those of ordinary skill in the art.

[0258] In general, the gene expression control sequence shall include, as

necessary, 5' non-transcribing and 5' non-translating sequences involved with the initiation of transcription and translation, respectively, such as a TATA box, capping sequence, CAAT sequence, and the like. Especially, such 5' non- transcribing sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined coding nucleic acid. The gene expression sequences optionally include enhancer sequences or upstream activator sequences as desired.

[0259] Viral vectors include, but are not limited to, nucleic acid sequences from the following viruses: retrovirus, such as Moloney murine leukemia virus, Harvey murine sarcoma virus, murine mammary tumor virus, and Rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyomaviruses; Epstein- Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors well-known in the art. Certain viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses, the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell line with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in Kriegler, M., Gene Transfer and Expression, A Laboratory Manual, W.H. Freeman Co., New York (1990) and Murry, E. J., Methods in Molecular Biology, Vol. 7, Humana Press, Inc., Cliffton, N.J. (1991).

[0260] In one embodiment, the virus is an adeno-associated virus, a double- stranded DNA virus. The adeno-associated virus can be engineered to be replication-deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hematopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion.

[0261] Other vectors include plasmid vectors. Plasmid vectors have been

extensively described in the art and are well-known to those of skill in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. In the last few years, plasmid vectors have been found to be particularly advantageous for delivering genes to cells in vivo because of their inability to replicate within and integrate into a host genome. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operably encoded within the plasmid. Some commonly used plasmids available from commercial suppliers include pBR322, pUC18, pUC19, various pcDNA plasmids, pRC/CMV, various pCMV plasmids, pSV40, and pBlueScript. Additional examples of specific plasmids include pcDNA3.1, catalog number V79020; pcDNA3.1/hygro, catalog number V87020; pcDNA4/myc-His, catalog number V86320; and pBudCE4.1, catalog number V53220, all from Invitrogen (Carlsbad, CA.). Other plasmids are well-known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using standard molecular biology techniques to remove and/or add specific fragments of DNA.

[0262] In one insect expression system that may be used to produce the proteins of the invention, Autographa californica nuclear polyhidrosis virus (AcNPV) is used as a vector to express the foreign genes. The virus grows in Spodoptera frugiperda cells. A coding sequence may be cloned into non-essential regions (for example, the polyhedron gene) of the virus and placed under control of an

ACNPV promoter (for example, the polyhedron promoter). Successful insertion of a coding sequence will result in inactivation of the polyhedron gene and production of non-occluded recombinant virus (i.e., virus lacking the

proteinaceous coat coded for by the polyhedron gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed, (see, e.g., Smith et al. (1983) J Virol 46:584; U.S. Pat. No. 4,215,051). Further examples of this expression system may be found in Ausubel et al., eds. (1989) Current Protocols in Molecular Biology, Vol. 2, Greene Publish. Assoc. & Wiley Interscience.

[0263] Another system which can be used to express the proteins of the invention is the glutamine synthetase gene expression system, also referred to as the "GS expression system" (Lonza Biologies PLC, Berkshire UK). This expression system is described in detail in U.S. Pat. No. 5,981,216.

[0264] In mammalian host cells, a number of viral based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, a coding sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo

recombination. Insertion in a non-essential region of the viral genome (e.g. , region El or E3) will result in a recombinant virus that is viable and capable of expressing peptide in infected hosts. See, e.g., Logan & Shenk (1984) Proc Natl Acad Sci USA 81 :3655). Alternatively, the vaccinia 7.5 K promoter may be used. See, e.g., Mackett et al. (1982) Proc Natl Acad Sci USA 79:7415; Mackett et al. (1984) J Virol 49:857; Panicali et al. (1982) Proc Natl Acad Sci USA 79:4927.

[0265] To increase efficiency of production, the polynucleotides can be designed to encode multiple units of the protein of the invention separated by enzymatic cleavage sites. The resulting polypeptide can be cleaved {e.g., by treatment with the appropriate enzyme) in order to recover the polypeptide units. This can increase the yield of polypeptides driven by a single promoter. When used in appropriate viral expression systems, the translation of each polypeptide encoded by the mR A is directed internally in the transcript; e.g., by an internal ribosome entry site, IRES. Thus, the polycistronic construct directs the transcription of a single, large polycistronic mRNA which, in turn, directs the translation of multiple, individual polypeptides. This approach eliminates the production and enzymatic processing of polyproteins and may significantly increase the yield of polypeptides driven by a single promoter.

[0266] Vectors used in transformation will usually contain a selectable marker used to identify transformants. In bacterial systems, this can include an antibiotic resistance gene such as ampicillin or kanamycin. Selectable markers for use in cultured mammalian cells include genes that confer resistance to drugs, such as neomycin, hygromycin, and methotrexate. The selectable marker may be an amplifiable selectable marker. One amplifiable selectable marker is the dihydro folate reductase (DHFR) gene. Simonsen C C et al. (1983) Proc Natl Acad Sci USA 80:2495-9. Selectable markers are reviewed by Thilly (1986) Mammalian Cell Technology, Butterworth Publishers, Stoneham, Mass., and the choice of selectable markers is well within the level of ordinary skill in the art.

[0267] Selectable markers may be introduced into the cell on a separate plasmid at the same time as the gene of interest, or they may be introduced on the same plasmid. If on the same plasmid, the selectable marker and the gene of interest may be under the control of different promoters or the same promoter, the latter arrangement producing a dicistronic message. Constructs of this type are known in the art (for example, U.S. Pat. No. 4,713,339). [0268] The expression vectors can encode for tags that permit easy purification of the recombinantly produced protein. Examples include, but are not limited to, vector pUR278 (Ruther et al. (1983) EMBO J 2ΛΊ91), in which coding sequences for the protein to be expressed may be ligated into the vector in frame with the lac z coding region so that a tagged fusion protein is produced; pGEX vectors may be used to express proteins of the invention with a glutathione S-transferase (GST) tag. These proteins are usually soluble and can easily be purified from cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The vectors include cleavage sites (thrombin or Factor Xa protease or PRESCISSION PROTEASE™ (Pharmacia, Peapack, N.J.)) for easy removal of the tag after purification.

[0269] The expression vector or vectors are then transfected or co-transfected into a suitable target cell, which will express the polypeptides. Transfection techniques known in the art include, but are not limited to, calcium phosphate precipitation (Wigler et al. (1978) Cell 14:725), electroporation (Neumann et al. (1982) EMBO J 1 :841), and liposome-based reagents. A variety of host-expression vector systems may be utilized to express the proteins described herein including both prokaryotic and eukaryotic cells. These include, but are not limited to,

microorganisms such as bacteria {e.g., E. coli) transformed with recombinant bacteriophage DNA or plasmid DNA expression vectors containing an appropriate coding sequence; yeast or filamentous fungi transformed with recombinant yeast or fungi expression vectors containing an appropriate coding sequence; insect cell systems infected with recombinant virus expression vectors {e.g., baculovirus) containing an appropriate coding sequence; plant cell systems infected with recombinant virus expression vectors {e.g. , cauliflower mosaic virus or tobacco mosaic virus) or transformed with recombinant plasmid expression vectors {e.g., Ti plasmid) containing an appropriate coding sequence; or animal cell systems, including mammalian cells {e.g., HEK 293, CHO, Cos, HeLa, HKB11, and BHK cells).

[0270] In one embodiment, the host cell is a eukaryotic cell. As used herein, a eukaryotic cell refers to any animal or plant cell having a definitive nucleus.

Eukaryotic cells of animals include cells of vertebrates, e.g., mammals, and cells of invertebrates, e.g., insects. Eukaryotic cells of plants specifically can include, without limitation, yeast cells. A eukaryotic cell is distinct from a prokaryotic cell, e.g., bacteria.

[0271] In certain embodiments, the eukaryotic cell is a mammalian cell. A

mammalian cell is any cell derived from a mammal. Mammalian cells specifically include, but are not limited to, mammalian cell lines. In one embodiment, the mammalian cell is a human cell. In another embodiment, the mammalian cell is a HEK 293 cell, which is a human embryonic kidney cell line. HEK 293 cells are available as CRL-1533 from American Type Culture Collection, Manassas, VA, and as 293-H cells, Catalog No. 11631-017 or 293-F cells, Catalog No. 11625-019 from Invitrogen (Carlsbad, Calif). In some embodiments, the mammalian cell is a PER.C6^® cell, which is a human cell line derived from retina. PER.C6^® cells are available from Crucell (Leiden, The Netherlands). In other embodiments, the mammalian cell is a Chinese hamster ovary (CHO) cell. CHO cells are available from American Type Culture Collection, Manassas, VA. {e.g., CHO-K1; CCL- 61). In still other embodiments, the mammalian cell is a baby hamster kidney (BHK) cell. BHK cells are available from American Type Culture Collection, Manassas, Va. {e.g., CRL-1632). In some embodiments, the mammalian cell is a HKB11 cell, which is a hybrid cell line of a HEK293 cell and a human B cell line. Mei et al, Mol. Biotechnol. 34(2): 165-78 (2006).

[0272] In one embodiment, a plasmid including a FVIII(X)-Fc fusion coding sequence, a VWF fragment-L-Fc fusion coding sequence, or both and a selectable marker, e.g. , zeocin resistance, are transfected into HEK 293 cells, for production of a chimeric protein.

[0273] In another embodiment, a plasmid including a FVIII-Fc fusion coding sequence, a VWF fragment-XTEN-L-Fc fusion coding sequence, or both and a selectable marker, e.g., zeocin resistance, are transfected into HEK 293 cells, for production of a chimeric protein.

[0274] In other embodiments, a plasmid including a FVIII(X)-Fc fusion coding sequence, a Fc coding sequence, or both and a selectable marker, e.g., zeocin resistance, are transfected into HEK 293 cells, for production of a chimeric protein.

[0275] In some embodiments, a first plasmid including a FVIII(X)-Fc fusion

coding sequence and a first selectable marker, e.g., a zeocin resistance gene, and a second plasmid including an Fc coding sequence or a VWF fragment-L-Fc coding sequence and a second selectable marker, e.g., a neomycin resistance gene, and a third plasmid including a protein convertase coding sequence and a third selectable marker, e.g., a hygromycin resistance gene, are cotransfected into HEK 293 cells, for production of the chimeric protein. The first and second plasmids can be introduced in equal amounts (i.e. , 1 : 1 molar ratio), or they can be introduced in unequal amounts.

[0276] In still other embodiments, a first plasmid including a FVIII-Fc fusion coding sequence and a first selectable marker, e.g., a zeocin resistance gene, and a second plasmid including a VWF fragment-XTEN-L-Fc coding sequence and a second selectable marker, e.g. , a neomycin resistance gene, and a third plasmid including a protein convertase coding sequence and a third selectable marker, e.g., a hygromycin resistance gene, are cotransfected into HEK 293 cells, for production of the chimeric protein. The first and second plasmids can be introduced in equal amounts (i.e., 1 : 1 molar ratio), or they can be introduced in unequal amounts.

[0277] In yet other embodiments, a first plasmid including a FVIII(X)-Fc fusion coding sequence and a first selectable marker, e.g., a zeocin resistance gene, and a second plasmid including a VWF fragment-XTEN-L-Fc coding sequence and a second selectable marker, e.g. , a neomycin resistance gene, and a third plasmid including a protein convertase coding sequence and a third selectable marker, e.g., a hygromycin resistance gene, are cotransfected into HEK 293 cells, for production of the chimeric protein. The first and second plasmids can be introduced in equal amounts (i.e., 1 : 1 molar ratio), or they can be introduced in unequal amounts.

[0278] In certain embodiments, a first plasmid, including a chimeric protein

encoding FVIII (with or without XTEN)-F1-L1-V-XTEN-L2-F2 coding sequence and a first selectable marker, e.g. , a zeocin resistance gene, and a second plasmid including a protein convertase coding sequence and a second selectable marker, e.g., a hygromycin resistance gene, are cotransfected into HEK 293 cells, for production of the chimeric protein. The promoters for the FVIII(X)-Fc coding sequence and the VWF-XTEN-Fc coding sequence can be different or they can be the same. [0279] In still other embodiments, transfected cells are stably transfected. These cells can be selected and maintained as a stable cell line, using conventional techniques known to those of skill in the art.

[0280] Host cells containing DNA constructs of the protein are grown in an

appropriate growth medium. As used herein, the term "appropriate growth medium" means a medium containing nutrients required for the growth of cells. Nutrients required for cell growth may include a carbon source, a nitrogen source, essential amino acids, vitamins, minerals, and growth factors. Optionally, the media can contain one or more selection factors. Optionally the media can contain bovine calf serum or fetal calf serum (FCS). In one embodiment, the media contains substantially no IgG. The growth medium will generally select for cells containing the DNA construct by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker on the DNA construct or co-transfected with the DNA construct. Cultured mammalian cells are generally grown in commercially available serum-containing or serum-free media (e.g., MEM, DMEM, DMEM/F12). In one embodiment, the medium is CD293 (Invitrogen, Carlsbad, CA.). In another embodiment, the medium is CD 17 (Invitrogen, Carlsbad, CA.). Selection of a medium appropriate for the particular cell line used is within the level of those ordinary skilled in the art.

[0281] In order to co-express the two polypeptide chains of the chimeric protein, the host cells are cultured under conditions that allow expression of both chains. As used herein, culturing refers to maintaining living cells in vitro for at least a definite time. Maintaining can, but need not include, an increase in population of living cells. For example, cells maintained in culture can be static in population, but still viable and capable of producing a desired product, e.g., a recombinant protein or recombinant fusion protein. Suitable conditions for culturing eukaryotic cells are well known in the art and include appropriate selection of culture media, media supplements, temperature, pH, oxygen saturation, and the like. For commercial purposes, culturing can include the use of any of various types of scale-up systems including shaker flasks, roller bottles, hollow fiber bioreactors, stirred-tank bioreactors, airlift bioreactors, Wave bioreactors, and others. [0282] The cell culture conditions are also selected to allow association of the

VWF fragment with the FVIII protein. Conditions that allow expression of the VWF fragment and/or the FVIII protein may include the presence of a source of vitamin K. For example, in one embodiment, stably transfected HEK 293 cells are cultured in CD293 media (Invitrogen, Carlsbad, CA) or OptiCHO media

(Invitrogen, Carlsbad, CA) supplemented with 4 mM glutamine.

[0283] In one aspect, the present invention is directed to a method of expressing, making, or producing the chimeric protein of the invention comprising a) transfecting a host cell comprising a polynucleotide encoding the chimeric protein and b) culturing the host cell in a culture medium under a condition suitable for expressing the chimeric protein, wherein the chimeric protein is expressed.

[0284] In further embodiments, the protein product containing the VWF fragment linked to an XTEN sequence or the FVIII protein linked to an XTEN sequence is secreted into the media. Media is separated from the cells, concentrated, filtered, and then passed over two or three affinity columns, e.g. , a protein A column and one or two anion exchange columns.

[0285] In certain aspects, the present invention relates to the chimeric protein produced by the methods described herein.

[0286] In vitro production allows scale-up to give large amounts of the desired altered polypeptides of the invention. Techniques for mammalian cell cultivation under tissue culture conditions are known in the art and include homogeneous suspension culture, e.g. in an airlift reactor or in a continuous stirrer reactor, or immobilized or entrapped cell culture, e.g. in hollow fibers, microcapsules, on agarose microbeads or ceramic cartridges. If necessary and/or desired, the solutions of polypeptides can be purified by the customary chromatography methods, for example gel filtration, ion-exchange chromatography, hydrophobic interaction chromatography (HIC, chromatography over DEAE-cellulose or affinity chromatography.

PHARMACEUTICAL COMPOSITION

[0287] Compositions containing the chimeric protein of the present invention may contain a suitable pharmaceutically acceptable carrier. For example, they may contain excipients and/or auxiliaries that facilitate processing of the active compounds into preparations designed for delivery to the site of action. [0288] The pharmaceutical composition can be formulated for parenteral administration (i.e. intravenous, subcutaneous, or intramuscular) by bolus injection. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multidose containers with an added preservative. The compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g. , pyrogen free water.

[0289] Suitable formulations for parenteral administration also include aqueous solutions of the active compounds in water-soluble form, for example, water- soluble salts. In addition, suspensions of the active compounds as appropriate oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, including, for example, sodium carboxymethyl cellulose, sorbitol and dextran. Optionally, the suspension may also contain stabilizers. Liposomes also can be used to encapsulate the molecules of the invention for delivery into cells or interstitial spaces. Exemplary pharmaceutically acceptable carriers are physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like. In some embodiments, the composition comprises isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride. In other embodiments, the compositions comprise

pharmaceutically acceptable substances such as wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the active ingredients.

[0290] Compositions of the invention may be in a variety of forms, including, for example, liquid (e.g., injectable and infusible solutions), dispersions, suspensions, semi-solid and solid dosage forms. The preferred form depends on the mode of administration and therapeutic application.

[0291] The composition can be formulated as a solution, micro emulsion,

dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the active ingredient in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active ingredient into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0292] The active ingredient can be formulated with a controlled-release

formulation or device. Examples of such formulations and devices include implants, transdermal patches, and microencapsulated delivery systems.

Biodegradable, biocompatible polymers can be used, for example, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for the preparation of such formulations and devices are known in the art. See e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0293] Injectable depot formulations can be made by forming microencapsulated matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the polymer employed, the rate of drug release can be controlled. Other exemplary

biodegradable polymers are polyorthoesters and polyanhydrides. Depot injectable formulations also can be prepared by entrapping the drug in liposomes or microemulsions.

[0294] Supplementary active compounds can be incorporated into the

compositions. In one embodiment, the chimeric protein of the invention is formulated with another clotting factor, or a variant, fragment, analogue, or derivative thereof. For example, the clotting factor includes, but is not limited to, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, prothrombin, fibrinogen, von Willebrand factor or recombinant soluble tissue factor (rsTF) or activated forms of any of the preceding. The clotting factor of hemostatic agent can also include anti-fibrino lytic drugs, e.g., epsilon-amino- caproic acid, tranexamic acid.

[0295] Dosage regimens may be adjusted to provide the optimum desired

response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. See, e.g., Remington's Pharmaceutical Sciences (Mack Pub. Co., Easton, Pa. 1980).

[0296] In addition to the active compound, the liquid dosage form may contain inert ingredients such as water, ethyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethylformamide, oils, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, and fatty acid esters of sorbitan.

[0297] Non-limiting examples of suitable pharmaceutical carriers are also

described in Remington's Pharmaceutical Sciences by E. W. Martin. Some examples of excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition can also contain pH buffering reagents, and wetting or emulsifying agents.

[0298] For oral administration, the pharmaceutical composition can take the form of tablets or capsules prepared by conventional means. The composition can also be prepared as a liquid for example a syrup or a suspension. The liquid can include suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats), emulsifying agents (lecithin or acacia), non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils), and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations can also include flavoring, coloring and sweetening agents. Alternatively, the composition can be presented as a dry product for constitution with water or another suitable vehicle.

[0299] For buccal administration, the composition may take the form of tablets or lozenges according to conventional protocols.

[0300] For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of a nebulized aerosol with or without excipients or in the form of an aerosol spray from a pressurized pack or nebulizer, with optionally a propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoromethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0301] The pharmaceutical composition can also be formulated for rectal

administration as a suppository or retention enema, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

[0302] In one embodiment, a pharmaceutical composition comprises a chimeric protein, the polynucleotide encoding the chimeric protein, the vector comprising the polynucleotide, or the host cell comprising the vector, and a pharmaceutically acceptable carrier. The FVIII protein in a chimeric protein has extended half-life compared to wild type FVIII protein or the corresponding FVIII protein without the VWF fragment. In one embodiment, wherein the half-life of the FVIII protein is extended at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, or at least about 12 times longer than wild type FVIII. In another embodiment, the half-life of Factor VIII is at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, at least about 28 hours, at least about 29 hours, at least about 30 hours, at least about 31 hours, at least about 32 hours, at least about 33 hours, at least about 34 hours, at least about 35 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours.

[0303] In some embodiments, the composition is administered by a route selected from the group consisting of topical administration, intraocular administration, parenteral administration, intrathecal administration, subdural administration and oral administration. The parenteral administration can be intravenous or subcutaneous administration.

[0304] In other embodiments, the composition is used to treat a bleeding disease or condition in a subject in need thereof. The bleeding disease or condition is selected from the group consisting of a bleeding coagulation disorder,

hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, bleeding in the illiopsoas sheath and any combinations thereof. In still other embodiments, the subject is scheduled to undergo a surgery. In yet other embodiments, the treatment is prophylactic or on- demand.

GENE THERAPY

[0305] A chimeric protein thereof of the invention can be produced in vivo in a mammal, e.g., a human patient, using a gene therapy approach to treatment of a bleeding disease or disorder selected from the group consisting of a bleeding coagulation disorder, hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis,

gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, and bleeding in the illiopsoas sheath would be therapeutically beneficial. In one embodiment, the bleeding disease or disorder is hemophilia. In another embodiment, the bleeding disease or disorder is hemophilia A. This involves administration of a suitable chimeric protein-encoding nucleic acid operably linked to suitable expression control sequences. In certain embodiment, these sequences are incorporated into a viral vector. Suitable viral vectors for such gene therapy include adenoviral vectors, lentiviral vectors, baculoviral vectors, Epstein Barr viral vectors, papovaviral vectors, vaccinia viral vectors, herpes simplex viral vectors, and adeno associated virus (AAV) vectors. The viral vector can be a replication-defective viral vector. In other embodiments, an adenoviral vector has a deletion in its El gene or E3 gene. When an adenoviral vector is used, the mammal may not be exposed to a nucleic acid encoding a selectable marker gene. In other embodiments, the sequences are incorporated into a non- viral vector known to those skilled in the art.

METHODS OF USING CHIMERIC PROTEIN

[0306] The present invention is directed to a method of using a chimeric protein described herein to prevent or inhibit endogenous VWF binding to a FVIII protein. The present invention is also directed to a method of using a chimeric protein having a FVIII protein linked to XTEN and an Ig constant region or a portion thereof.

[0307] One aspect of the present invention is directed to preventing or inhibiting

FVIII interaction with endogenous VWF by blocking or shielding the VWF binding site on the FVIII from endogenous VWF and at the same time extending half-life of the FVIII protein using an XTEN sequence in combination with an Ig constant region or a portion thereof, which can also be a half-life extender. In one embodiment, the invention is directed to a method of constructing a FVIII protein having half-life longer than wild-type FVIII. In one embodiment, an XTEN sequence inhibits or prevents interaction of a FVIII protein in a chimeric protein with endogenous VWF. In another embodiment, an Ig constant region or a portion thereof inhibits or prevents interaction of the FVIII protein with endogenous VWF. The chimeric protein useful in the method includes any one or more chimeric protein described herein.

[0308] Another aspect of the invention includes a method of administering to a subject in need thereof a chimeric protein comprising a FVIII protein having half- life longer than wild-type FVIII, wherein the method comprises administering the chimeric protein described herein to the subject.

[0309] In one embodiment, the invention is directed to a method of using an

XTEN sequence and an Ig constant region or a portion thereof to extend a half-life of a FVIII protein and a VWF fragment to prevent or inhibit endogenous VWF interaction with a FVIII protein. A FVIII protein linked to an XTEN sequence (e.g., FVIII(X)) and then bound to or associated with a VWF fragment is shielded or protected from the clearance pathway of VWF and thus has reduced clearance compared to the FVIII protein not bound to the VWF fragment. The shielded FVIII protein thus has maximum extension of a half-life compared to a FVIII protein not bound to or associated with the XTEN sequence and the VWF fragment. In certain embodiments, the FVIII protein associated with or protected by a VWF fragment and linked to an XTEN sequence is not cleared by a VWF clearance receptor. In other embodiments, the FVIII protein associated with or protected by a VWF fragment and linked to an XTEN sequence is cleared from the system slower than the FVIII protein that is not associated with or protected by the VWF fragment and linked to the XTEN sequence.

[0310] In one aspect, the chimeric protein comprising the FVIII protein linked to an XTEN sequence or the FVIII protein bound to or associated with a VWF fragment linked to XTEN has reduced clearance from circulation as the VWF fragment does not contain a VWF clearance receptor binding site. The VWF fragment prevents or inhibits clearance of FVIII bound to or associated with the VWF fragment from the system through the VWF clearance pathway. The VWF fragments useful for the present invention can also provide at least one or more VWF-like FVIII protection properties that are provided by endogenous VWF. In certain embodiments, the VWF fragment or the XTEN sequence can also mask one or more FVIII clearance receptor binding site, thereby preventing clearance of FVIII by its own clearance pathway.

[0311] In some embodiments, the prevention or inhibition of a FVIII protein

binding to endogenous VWF by the VWF fragment or the XTEN sequence can be in vitro or in vivo.

[0312] Also provided is a method of increasing the half-life of a FVIII protein comprising administering the chimeric protein described herein to a subject in need thereof. The half-life of non-activated FVIII bound to or associated with full-length VWF is about 12 to 14 hours in plasma. In VWD type 3, wherein there is almost no VWF in circulation, the half-life of FVIII is only about six hours, leading to symptoms of mild to moderate hemophilia A in such patients due to decreased concentrations of FVIII. The half-life of the FVIII protein linked to or associated with the VWF fragment or the XTEN sequence of the present invention can increase at least about 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.6 times, 2.7. times, 2.8 times, 2.9 times, 3.0 times, 3.1 times, 3.2 times, 3.3 times, 3.4 times, 3.5 times, 3.6 times, 3.7 times, 3.8 times, 3.9 times, or 4.0 times higher than the half-life of the non- activated FVIII bound to or associated with full-length VWF.

[0313] In one embodiment, the half-life of the FVIII protein linked to or

associated with the VWF fragment or linked to an Ig constant region or a portion thereof in the chimeric protein comprising an XTEN sequence increases at least about 2 times, 2.5 times, 3.0 times, 3.5 times, 4.0 times, 4.5 times, 5.0 times, 5.5 times, 6.0 times, 7 times, 8 times, 9 times, or 10 times higher than the half-life of the non-activated FVIII bound to or associated with full-length VWF. In another embodiment, the half-life of the FVIII protein linked to or associated with the VWF fragment or an Ig constant region or a portion thereof in the chimeric protein comprising an XTEN sequence increases about 2 to about 5 times, about 3 to about 10 times, about 5 to about 15 times, about 10 to about 20 times, about 15 to about 25 times, about 20 to about 30 times, about 25 to about 35 times, about 30 to about 40 times, about 35 to about 45 times higher than the half-life of the non- activated FVIII bound to or associated with full-length VWF or wild type FVIII. In a specific embodiment, the half-life of the FVIII protein linked to or associated with the VWF fragment or linked to an Ig constant region in the chimeric protein comprising an XTEN sequence increases at least about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 times higher than the half-life of the wild type FVIII in a FVIII and VWF double knockout mouse.

[0314] In some embodiments, the half-life of the chimeric protein comprising the

VWF fragment fused to a first Ig constant region or a portion thereof, e.g., a first Fc region and an XTEN sequence, and a FVIII protein linked to an XTEN sequence and a second Ig constant region or a portion thereof, e.g., a second Fc region, is longer than the half-life of a FVIII associated with endogenous VWF. In other embodiments, the half-life of the chimeric protein is at least about 1.5 times, 2 times, 2.5 times, 3.5 times, 3.6 times, 3.7 times, 3.8 times, 3.9 times, 4.0 times, 4.5 times, or 5.0 times the half-life of wild type FVIII or a FVIII protein associated with endogenous VWF. In some embodiments, as a result of the invention the half-life of the FVIII protein is extended compared to a FVIII protein without the VWF fragment or wild-type FVIII. The half-life of the chimeric protein of the invention is at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, or at least about 12 times longer than the half-life of a FVIII protein without the VWF fragment or wild-type FVIII. In one embodiment, the half-life of FVIII is about 1.5 -fold to about 20-fold, about 1.5 fold to about 15 fold, or about 1.5 fold to about 10 fold longer than the half-life of wild-type FVIII. In another embodiment, the half-life of the FVIII is extended about 2-fold to about 10-fold, about 2-fold to about 9-fold, about 2-fold to about 8-fold, about 2-fold to about 7-fold, about 2-fold to about 6-fold, about 2-fold to about 5-fold, about 2- fold to about 4-fold, about 2-fold to about 3-fold, about 2.5-fold to about 10-fold, about 2.5-fold to about 9-fold, about 2.5-fold to about 8-fold, about 2.5-fold to about 7-fold, about 2.5-fold to about 6-fold, about 2.5-fold to about 5-fold, about 2.5 -fold to about 4-fold, about 2.5 -fold to about 3 -fold, about 3 -fold to about 10- fold, about 3-fold to about 9-fold, about 3-fold to about 8-fold, about 3-fold to about 7-fold, about 3-fold to about 6-fold, about 3-fold to about 5-fold, about 3- fold to about 4-fold, about 4-fold to about 6 fold, about 5 -fold to about 7-fold, or about 6-fold to about 8 fold as compared to wild-type FVIII or a FVIII protein without the VWF fragment. In other embodiments, the half-life of the chimeric protein of the invention is at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, at least about 28 hours, at least about 29 hours, at least about 30 hours, at least about 31 hours, at least about 32 hours, at least about 33 hours, at least about 34 hours, at least about 35 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours. In still other embodiments, the half-life of the chimeric protein of the invention is about 15 hours to about two weeks, about 16 hours to about one week, about 17 hours to about one week, about 18 hours to about one week, about 19 hours to about one week, about 20 hours to about one week, about 21 hours to about one week, about 22 hours to about one week, about 23 hours to about one week, about 24 hours to about one week, about 36 hours to about one week, about 48 hours to about one week, about 60 hours to about one week, about 24 hours to about six days, about 24 hours to about five days, about 24 hours to about four days, about 24 hours to about three days, or about 24 hours to about two days.

[0316] In some embodiments, the average half-life of the chimeric protein of the invention per subject is about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours (1 day), about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 40 hours, about 44 hours, about 48 hours (2 days), about 54 hours, about 60 hours, about 72 hours (3 days), about 84 hours, about 96 hours (4 days), about 108 hours, about 120 hours (5 days), about six days, about seven days (one week), about eight days, about nine days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days.

[0317] In addition, the invention provides a method of treating or preventing a bleeding disease or disorder comprising administering an effective amount of a chimeric protein. In one embodiment, the bleeding disease or disorder is selected from the group consisting of a bleeding coagulation disorder, hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinal bleeding, intracranial hemorrhage, intraabdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the

retroperitoneal space, and bleeding in the illiopsoas sheath. In a specific embodiment, the bleeding disease or disorder is hemophilia A.

[0318] The chimeric protein comprising an XTEN sequence and an Ig constant region or a portion thereof in combination with a VWF fragment described herein, that prevents or inhibits interaction of the FVIII protein with endogenous VWF prepared by the invention, has many uses as will be recognized by one skilled in the art, including, but not limited to methods of treating a subject having a hemostatic disorder and methods of treating a subject in need of a general hemostatic agent. In one embodiment, the invention relates to a method of treating a subject having a hemostatic disorder comprising administering a therapeutically effective amount of the chimeric protein.

[0319] The FVIII protein portion in the chimeric protein treats or prevents a

hemostatic disorder by serving as a cofactor to Factor IX on a negatively charged phospholipid surface, thereby forming a Xase complex. The binding of activated coagulation factors to a phospholipid surface localizes this process to sites of vascular damage. On a phospholipid surface, Factor Villa increases the maximum velocity of Factor X activation by Factor IXa, by approximately 200,000-fold, leading to the large second burst of thrombin generation.

[0320] The chimeric protein of the invention can be used to treat any hemostatic disorder. The hemostatic disorders that may be treated by administration of the chimeric protein of the invention include, but are not limited to, hemophilia A, as well as deficiencies or structural abnormalities relating to Factor VIII. In one embodiment, the hemostatic disorder is hemophilia A.

[0321] The chimeric protein of the invention can be used prophylactically to treat a subject with a hemostatic disorder. The chimeric protein of the invention can be used to treat an acute bleeding episode in a subject with a hemostatic disorder. In another embodiment, the hemostatic disorder can be the result of a defective clotting factor, e.g., von Willebrand's factor. In one embodiment, the hemostatic disorder is an inherited disorder. In another embodiment, the hemostatic disorder is an acquired disorder. The acquired disorder can result from an underlying secondary disease or condition. The unrelated condition can be, as an example, but not as a limitation, cancer, an auto-immune disease, or pregnancy. The acquired disorder can result from old age or from medication to treat an underlying secondary disorder {e.g. cancer chemotherapy).

[0322] The invention also relates to methods of treating a subject that does not have a congenital hemostatic disorder, but has a secondary disease or condition resulting in acquisition of a hemostatic disorder, e.g., due to development of an anti-FVIII antibody or a surgery. The invention thus relates to a method of treating a subject in need of a general hemostatic agent comprising administering a therapeutically effective amount of the chimeric protein prepared by the present methods. [0323] The present invention is also related to methods of reducing

immunogenicity of FVIII or inducing less immunogenicity against FVIII comprising administering an effective amount of the chimeric proteins described herein, or the polynucleotides encoding the same.

[0324] In one embodiment, the subject in need of a general hemostatic agent is undergoing, or is about to undergo, surgery. The chimeric protein of the invention can be administered prior to, during, or after surgery as a prophylactic regimen. The chimeric protein of the invention can be administered prior to, during, or after surgery to control an acute bleeding episode..

[0325] The chimeric protein of the invention can be used to treat a subject having an acute bleeding episode who does not have a hemostatic disorder. The acute bleeding episode can result from severe trauma, e.g., surgery, an automobile accident, wound, laceration gun shot, or any other traumatic event resulting in uncontrolled bleeding. Non limiting examples of bleeding episodes include a bleeding coagulation disorder, hemarthrosis, muscle bleed, oral bleed,

hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, bleeding in the illiopsoas sheath, and any combinations thereof.

[0326] In prophylactic applications, one or more compositions containing the chimeric protein of the invention or a cocktail thereof are administered to a patient not already in the disease state to enhance the patient's resistance or reduce symptoms associated with a disease or disorder. Such an amount is defined to be a "prophylactic effective dose." In therapeutic applications, a relatively high dosage {e.g., from about 1 to 400 mg/kg of polypeptide per dose, with dosages of from 5 to 25 mg being more commonly used for radioimmuno conjugates and higher doses for cytotoxin-drug modified polypeptides) at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.

[0327] In some embodiments, a chimeric protein or a composition of the invention is used for on-demand treatment, which includes treatment for a bleeding episode, hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis (head trauma), gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, or bleeding in the illiopsoas sheath. The subject may be in need of surgical prophylaxis, peri-operative management, or treatment for surgery. Such surgeries include, e.g., minor surgery, major surgery, tooth extraction, tonsillectomy, inguinal herniotomy, synovectomy, total knee replacement, craniotomy, osteosynthesis, trauma surgery, intracranial surgery, intra-abdominal surgery, intrathoracic surgery, or joint replacement surgery.

[0328] In one embodiment, the chimeric protein of the present invention is

administered intravenously, subcutaneously, intramuscularly, or via any mucosal surface, e.g., orally, sublingually, buccally, nasally, rectally, vaginally or via pulmonary route. The chimeric protein comprising a VWF fragment and a FVIII protein of the present invention can be implanted within or linked to a biopolymer solid support that allows for the slow release of the chimeric protein to the site of bleeding or implanted into bandage/dressing. The dose of the chimeric protein will vary depending on the subject and upon the particular route of administration used. Dosages can range from 0.1 to 100,000 μg/kg body weight. In one embodiment, the dosing range is 0.1-1,000 μg/kg. In another embodiment, the dosing range is 0.1-500 μg/kg. The protein can be administered continuously or at specific timed intervals. In vitro assays may be employed to determine optimal dose ranges and/or schedules for administration. In vitro assays that measure clotting factor activity are known in the art, e.g., STA-CLOT VIIa-rTF clotting assay or ROTEM clotting assay. Additionally, effective doses may be

extrapolated from dose-response curves obtained from animal models, e.g., a hemophiliac dog (Mount et al. 2002, Blood 99(8):2670).

[0329] Having now described the present invention in detail, the same will be more clearly understood by reference to the following examples, which are included herewith for purposes of illustration only and are not intended to be limiting of the invention. All patents, publications, and articles referred to herein are expressly and specifically incorporated herein by reference. Examples

[0330] Throughout the examples, the following materials and methods were used unless otherwise stated.

Materials and Methods

[0331] In general, the practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, biophysics, molecular biology, recombinant DNA technology, immunology (especially, e.g., antibody technology), and standard techniques in electrophoresis. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: Cold Spring Harbor Laboratory Press (1989); Antibody Engineering Protocols (Methods in Molecular Biology), 510, Paul, S., Humana Pr (1996); Antibody Engineering: A Practical Approach (Practical Approach Series, 169), McCafferty, Ed., Irl Pr (1996); Antibodies: A Laboratory Manual, Harlow et al., CS.H.L. Press, Pub. (1999); and Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons (1992). Example 1 : Cloning different VWF domains (Figure 1)

(a) Cloning of pSYN-VWF-002

[0332] pSYN-VWF-002 contains nucleotide sequences encoding a VWF

fragment, which are amino acids 1-477 of SEQ ID NO: 100. [VWF-DO3 protein sequence] Amino acid numbering represents the mature VWF sequence without propeptide and corresponds to amino acids 764-1240 of SEQ ID NO: 2. pSYN- VWF-002 construct has the FVIII signal peptide at N-terminus, which allows proper secretion of the synthesized protein and followed by a 6xHis tag at C- terminus, which is used for protein purification. It was synthesized by using following primer combinations:

ESC48- Fwd - VWF-DO3 with VIII signal and BsiWl site

TCGCGACGTACGGCCGCCACCATGCAAATAGAGCTCTCCACCTGCT TCT T TCTGT GCCT T T TGCGAT TCTGCT T TAGCCTATCCTGTCGGCCCCCCATG (SEQ ID NO: 90)

ESC51- Rev- VWF D^*D3 (1-477 amino acid) with 6His and Not 1 site

TGACCTCGAGCGGCCGCTCAGTGGTGATGGTGATGATGCGGCTCCTGGCAGGCT T CACAGGTGAGGT TGACAAC (SEQ ID NO: 91)

[0333] A 50 μΐ PCR reaction was carried out with ESC 48/ESC 51 primer

combinations and full length VWF plasmid as the template, using the 2 step PCR amplification cycle: 94 °C 2minutes; 21 cycles of (96 °C 30 seconds, 68 °C 2 minute). The 1460bp band was gel purified with a Gel Extraction kit (Qiagen, Valencia, Calif.) and cloned into the BsiWI and Notl restriction sites of pcDNA 4 to generate pSYN-VWF 002.

(b) Cloning of pSYN-VWF- 010 and 013

[0334] pS YN-VWF-010 was constructed using pS YN-VWF-008 and pS YN-

VWF-002. pSYN-VWF-008 contains the full-length VWF sequence in pcDNA 3.1 (amino acids 1-2813 of SEQ ID NO: 2), it includes 763 amino acid propeptide (i.e., D1D2 domains) followed by remaining 2050 amino acids sequence of mature VWF. The FVIII signal peptide in pSYN-VWF-002 was replaced with D1D2 domains from pS YN-VWF-008, the resulting construct is pSYN-VWF-010.

pSYN-VWF- 008 has a BamHl site at Arg907 and Notl site at the end of coding region (after stop codon). pSYN- VWF- 008 and 002 were digested with BamHl and Notl restriction enzymes. Inserts from pSYN- VWF-002 (1026 bp) were ligated into bamHl/Notl digested pSYN-VWF- 008 (8242bp) to obtain pSYN- VWF -010 (D1D2DO3: amino acid 1-1240 of SEQ ID NO: 2), a 6xHis tag was also added at the C-terminus. In transformed cells pS YN-VWF-010 is synthesized with propeptide but due to intracellular processing the secreted products do not contain any propeptide (D1D2). Protein from VWF-010 exists as dimer.

[0335] pS YN-VWF-010 was used to generate pS YN-VWF-013 which has two point mutations at C336A and C379A corresponding to SEQ ID NO: 100 (amino acid numbering represents mature VWF sequence without D1D2 domain-VWF sequence 2). These mutations are predicted to prevent dimerization of VWF D'D3 domain.

(c) Cloning of pSYN-VWF-025 and pSYN-VWF-029

[0336] pSYN-VWF-025 contains wild type D1D2D^*D3 sequences of full-length

VWF in pLIVE vector, and pSYN-VWF-029 contains D1D2DO3 sequence with C336A and C379A mutation. For cloning pSYN-VWF-025, the following primer combination was used:

ESC 89-fwd with Nhelsite= CTCACTATAGGGAGACCCAAGCTGGCTAGCCG

(SEQ ID NO: 92) ESC 91-rev with Sall=

CTGGATCCCGGGAGTCGACTCGTCAGTGGTGATGGTGATGATG (SEQ ID NO: 93)

[0337] A 50 μΐ PCR reaction was carried out with ESC 89/ESC91 primer

combinations and either pSYN-VWF 010 ( for pSYN-VWF-025) or pSYN-VWF 013 ( for pSYN-VWF-029) plasmid as the template using the 3 step PCR amplification cycle: 94 °C 2minutes; 21 cycles of (96 °C -30 seconds, 55 °C-30 second, 68 °C-4 minutes). The expected sized band (~3800bp) was gel purified with a Gel Extraction kit (Qiagen, Valencia, Calif.) and cloned into the Nheland Sail restriction sites of pLIVE-Mirus vector (Invitrogen, Carlsbad, Calif.) to generate pSYN-VWF 025 and 029.

(d) Cloning pSYN-VWF-031

[0338] pSYN-VWF-031 is a D1D2D'D3(C336A/C379A) -Fc construct which has a 48 amino acid long thrombin cleavable linker (8x GGGGS (SEQ ID NO 94) + thrombin site) in between the VWF D1D2D^*D3(C336A/C379A) and the Fc sequences. To make this construct, VWF-Fc region was amplified from construct pSYN-FVIII-064 (refer FVIII-VWF construct below). pSYN-F VIII- VWF was digested with Xbal and Nhel . Resulting insert region of 4165bp, containing the

VWF fragment and Fc region was used as a template for amplifying the VWF and

Fc region by primer combinations LW 22/LW23.

LW 22-FWD-VWF-D^*D3 with FVIII signal sequence and BsiWl site

GCGCCGGCCGTACGATGCAAATAGAGCTCTCCACCTGCT TCT T TCTGTGCCT T T T GCGAT TCTGCT T TAGCCTATCCTGTCGGCCCCCCATG (SEQ ID NO: 95) LW 23-Rev- Fc with stop codon and Notl site

TCATCAATGTATCT TATCATGTCTGAAT TCGCGGCCGCTCAT T TACC (SEQ ID NO:96)

[0339] The PCR product obtained from LW22/LW23 amplification (~2300bp) was cloned in BsiWl/Notl digested pSYN-VWF-002 to obtain pSYN-VWF-014 intermediate. pSYN-VWF-014 contains FVIII signal peptide -D'D3 -20 amino acid thrombin cleavable linker followed by the Fc region.

[0340] To generate the D1D2D'D3-Fc construct, the D1D2DO3 region was amplified from pSYN-VWF-013 using primer combination LW24/LW27 by standard PCR method.

LW24- Fwd- VWF D1D2DO3 cloning oligo with BsiWl site GCGCCGGCCGTACGATGATTCCTGCCAGATTTGCCGGGGTG (SEQ ID NO:97) LW27-Rev-VWF D^*D3 oligo with EcoRV

CCACCGCCAGATATCGGCTCCTGGCAGGCTTCACAGGTGAG (SEQ ID NO:98)

[0341] The PCR product obtained from LW22/LW23 amplification (~3750bp) was cloned in BsiWl/EcoRV digested pSYN-VWF-014 to obtain pSYN-VWF- 015 intermediate. The linker length between the VWF fragment and Fc region was changed to obtain pSYN-VWF-031.

VWF-D1D2DO3 protein sequence 1 (SEQ ID NO: 99)

1 MI PARFAGVL LALALILPGT LCAEGTRGRS STARCSLFGS

DFVNTFDGSM

51 YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE

FFDIHLFVNG

101 TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGFVARI

DGSGNFQVLL

151 SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL TSDPYDFANS

WALSSGEQWC

201 ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL

VDPEPFVALC

251 EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA

CSPVCPAGME

301 YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG

LCVESTECPC

351 VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV

TGQSHFKSFD

401 NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC

TRSVTVRLPG

451 LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL RIQHTVTASV

RLSYGEDLQM

501 DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG

LAEPRVEDFG

551 NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP

TFEACHRAVS

601 PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV

AWREPGRCEL

651 NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP

PGLYMDERGD

701 CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGFMHCTM

SGVPGSLLPD

751 AVLSSPLSHR SKRSLSCRPP MVKLVCPADN LRAEGLECTK

TCQNYDLECM

801 SMGCVSGCLC PPGMVRHENR CVALERCPCF HQGKEYAPGE

TVKIGCNTCV

851 CRDRKWNCTD HVCDATCSTI GMAHYLTFDG LKYLFPGECQ

YVLVQDYCGS

901 NPGTFRILVG NKGCSHPSVK CKKRVTILVE GGEIELFDGE

VNVKRPMKDE 951 THFEVVESGR YIILLLGKAL SVVWDRHLSI SVVLKQTYQE

KVCGLCGNFD

1001 GIQNNDLTSS NLQVEEDPVD FGNSWKVSSQ CADTRKVPLD

SSPATCHNNI

1051 MKQTMVDSSC RILTSDVFQD CNKLVDPEPY LDVCIYDTCS

CESIGDCACF

1101 CD IAAYAHV CAQHGKVVTW RTATLCPQSC EERNLRENGY

ECEWRYNSCA

1151 PACQVTCQHP EPLACPVQCV EGCHAHCPPG KILDELLQTC

VDPEDCPVCE

1201 VAGRRFASGK KVTLNPSDPE HCQICHCDVV NLTCEACQEP*

VWF-D ' D3 protein sequence 2 (SEQ ID NO: 100) 1 SLSCRPPMVK LVCPADNLRA EGLECTKTCQ NYDLECMSMG CVSGCLCPPG

51 MVRHENRCVA LERCPCFHQG KEYAPGETVK IGCNTCVCRD

RKWNCTDHVC

101 DATCSTIGMA HYLTFDGLKY LFPGECQYVL VQDYCGSNPG

TFRILVGNKG

151 CSHPSVKCKK RVTILVEGGE IELFDGEVNV KRPMKDETHF

EVVESGRYII

201 LLLGKALSVV WDRHLSISVV LKQTYQEKVC GLCGNFDGIQ

NNDLTSSNLQ

251 VEEDPVDFGN SWKVSSQCAD TRKVPLDSSP ATCHNNIMKQ

TMVDSSCRIL

301 TSDVFQDCNK LVDPEPYLDV CIYDTCSCES IGDCACFCDT

IAAYAHVCAQ

351 HGKVVTWRTA TLCPQSCEER NLRENGYECE WRYNSCAPAC

QVTCQHPEPL

401 ACPVQCVEGC HAHCPPGKIL DELLQTCVDP EDCPVCEVAG

RRFASGKKVT

451 LNPSDPEHCQ ICHCDVVNLT CEACQEP

Example 2: Effects of D'D3 and XTEN fusion on FVIII half-life extension

[0342] To evaluate D'D3 FVIII half-life extension potential on rFVIII-XTEN

fusion protein, a VWF D'D3 dimer was introduced into FVIII-VWF DKO mice by hydrodynamic injection of its corresponding DNA construct VWF-025 (Example 1). After D'D3 has reached the steady state expression (day5 post injection), a single dose of rFVIII-XTEN was administered by IV injection at 200 IU/kg dose. Blood samples were collected up to 120hrs post rFVIII-XTEN dosing. Plasma FVIII activity was analyzed by a FVIII chromogenic assay. The D'D3 expression level was measured by VWF ELISA, and rFVIIIFc PK profile was analyzed using WinNonlin program.

[0343] The study results were shown in Figure 2, and the PK parameter of rFVIII-

XTEN with/without D'D3 in circulation was listed in Table 16. The D'D3 dimer further extended rFIII-XTEN ti/₂ from 3.4hr to 17.8hr, a 5 fold increase. In addition to half-life, 5 fold of increase on MRT, 3.6 fold increases on AUC, 3.8 fold decreases on clearance were also observed.

[0344] We have observed a synergistic effect of D'D3 fragment and XTEN

technology, a serial of FVIII/VWF/XTEN constructs will be evaluated for their FVIII half-life extension potential in Hemophilic animals.

TABLE 16: rFVIII-XTEN PK parameter with/without D'D3 in blood circulation

Protein purification of FVIII-XTEN

[0345] An AE288 XTEN was inserted at the C-terminus of BDD-FVIII for this study. To purify this protein, a tangential flow filtration (TFF) step was used first to buffer exchange the conditioned media. Products in the filtrate were then captured using a strong anion exchange chromatography, and then further purified using affinity chromatography. Purity of the molecule was acceptable by HPLC- SEC and was further confirmed by western blotting. The specific activity of the molecule was comparable to B-domain deleted FVIII, as measured by aPTT assay and ELISA.

FVIII chromogenic assay

[0346] The FVIII activity was measured using the COATEST SP FVIII kit from

DiaPharma (lot# N089019) and all incubations were performed on a 37°C plate heater with shaking.

[0347] The range of rFVIII standard was from 100 mlU/mL to 0.78 mlU/mL. A pooled normal human plasma assay control and plasma samples (diluted with IX Coatest buffer) were added into Immulon 2HB 96-well plates in duplicate (25 μΕΛνεΙΙ). Freshly prepared IXa/FX Phospholipid mix (50 μί), 25 of 25mM CaC12, and 50 μΐ, of FXa substrate were added sequentially into each well with 5 minutes incubation between each addition. After incubating with the substrate, 25 μΐ_^ of 20% Acetic Acid was added to terminate the color reaction, and the absorbance of OD405 was measured with a SpectraMAX plus (Molecular Devices) instrument. Data were analyzed with SoftMax Pro software (version 5.2). The Lowest Level of Quantification (LLOQ) is 7.8 mlU/mL.

VWF ELBA:

[0348] Goat anti-human VWF antibody (Affinity purified, affinity biological,

GAVWF-AP) was used as the capture antibody at 0.5ug/well and VWF-EIA-D (Affinity Biologicals, VWF-EIA-D, 1 : 100 dilution) was used as the detecting antibody for the VWF ELISA. ELIS A assay was performed following the standard ELISA procedure, TMB was used as the HRP substrate, PBST/1.5% BSA/0.5M NaCl buffer was used as blocking and binding buffer. The assay standard range is lOOng to 0.78ng, and assay's lowest limit of quantification (LLOQ) is 7.8ng/mL.

Example 3: Plasmid construction of XTEN containing FVIII/VWF constructs (a) Cloning of pSYN-FVIII-161 (Figure 3)

[0349] The FVIII-161 plasmid comprises a single chain Fc (scFc) scaffold with enzyme cleavage sites which are processed during synthesis in a cell. The construct has a FVIII binding domain of full-length VWF ( D'D3).

[0350] Plasmid (pSYN-FVIII-161) was designed for the expression FVIII-Fc and

VWF-Fc heterodimer, where the D'D3 domains to bind FVIII and prevents FVIII interaction with phospholipids and activated protein C. Protein from pSYN-FVIII- 161 is expressed in the cell as a single polypeptide where the C-terminus of the FVIII-Fc subunit is linked to the N-terminus of the VWF D'D3-Fc subunit by a 6x (GGGGS) polypeptide linker (SEQ ID NO: 64). In addition, RR RS (SEQ ID NO: 11) and R RR R (SEQ ID NO: 10) sequences were inserted at the 5^* and 3^* end of the polypeptide linker, respectively, for intracellular cleavage by proprotein convertases following the last Arg at each sequence. Hence, the cells can express a double chain FVIII-Fc/D'D3-Fc heterodimer where the FVIII-Fc chain has a RRRRS sequence (SEQ ID NO: 11) at the C-terminus, but the remainder of the linker sequence has been removed. An AE288 XTEN fragment immediately followed by IS {5X(GGGGS)}LVPRGSGG (SEQ ID NO: 122) polypeptide (contains thrombin cleavage site) is introduced in between the VWF domains and the Fc region to facilitate release of the VWF fragment from FVIII once the FVIII-

VWF hetero-dimeric protein is activated by thrombin allowing interaction of

FVIII with other clotting factors.

pSYN-FVIII-161 (SEQ ID NO: 101).protein sequence (FVIII sequence amino acid position 1-1457; underlined region represents Fc region; curvy underline represents cleavable linker in between first Fc and VWF fragment; double underlined region represents VWF fragment; bold region represents cleavable linker in between VWF fragment and Fc.

1 MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL

GELPVDARFP

51 PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL

LGPTIQAEVY

101 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR

EKEDDKVFPG

151 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG

LIGALLVCRE

201 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD

AASARAWPKM

251 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL

EGHTFLVRNH

301 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME

AYVKVDSCPE

351 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI

RSVAKKHPKT

401 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR

KYKKVRFMAY

451 TDETFKTREA IQHESGILGP LLYGEVGDTL LI IFKNQASR

PYNIYPHGIT

501 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP

TKSDPRCLTR

551 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR

NVILFSVFDE

601 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHS INGYV

FDSLQLSVCL

651 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF

PFSGETVFMS

701 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED

SYEDI SAYLL

751 SKNNAIEPRS FSQNPPVLKR HQREITRTTL QSDQEEIDYD

D ISVEMKKE

801 DFDIYDEDEN QSPRSFQKKT RHYFIAAVER LWDYGMSSSP

HVLRNRAQSG

851 SVPQFKKVVF QEFTDGSFTQ PLYRGELNEH LGLLGPYIRA

EVED IMVTF

901 RNQASRPYSF YSSLISYEED QRQGAEPRKN FVKPNETKTY

FWKVQHHMAP 951 TKDEFDCKAW AYFSDVDLEK DVHSGLIGPL LVCHTNTLNP AHGRQVTVQE

1001 FALFF IFDE TKSWYFTENM ERNCRAPCNI QMEDPTFKEN YRFHAINGYI

1051 MDTLPGLVMA QDQRIRWYLL SMGSNENIHS IHFSGHVFTV RKKEEYKMAL

1101 YNLYPGVFET VEMLPSKAGI WRVECLIGEH LHAGMSTLFL VYSNKCQTPL

1151 GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK EPFSWIKVDL

1201 LAPMI IHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY RGNSTGTLMV

1251 FFGNVDSSGI KHNIFNPPII ARYIRLHPTH YSIRSTLRME LMGCDLNSCS

1301 MPLGMESKAI SDAQITASSY FTNMFATWSP SKARLHLQGR SNAWRPQVNN

1351 PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM YVKEFLISSS QDGHQWTLFF

1401 QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH QIALRMEVLG

1451 CEAQDLYDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV

1501 VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV

SVLTVLHQDW

5 I LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP

SRDELTKNQV

)1 SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS

FFLYSKLTVD

1651 KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGKRRRRSG GGG^GGGGSG

1701 GGGSGGGGSG GGGSGGGGSR KRRKRSLSCR PPMVKLVCPA DNLRAEGLEC

1751 TKTCONYDLE CMSMGCVSGC LCPPGMVRHE NRCVALERCP CFHQGKEYAP

1801 GETVKIGCNT CVCRDRKWNC TDHVCDATCS TIGMAHYLTF DGLKYLFPGE

1.851 CQYVLVQDYC GSNPGTFRIL VGNKGCSHPS VKCKKRVTIL VEGGEIELFD

1901 GEVNVKRPMK DETHFEVVES GRYI ILLLGK ALSVVWDRHL SISVVLKQTY

1951 QEKVCGLCGN FDGIQNNDLT SSNLQVEEDP VDFGNSWKVS SOCADTRKVP

2001 LDSSPATCHN NIMKQTMVDS SCRILTSDVF QDCNKLVDPE PYLDVCIYDT

2051 CSCESIGDCA AFCDTIAAYA HVCAQHGKVV TWRTATLCPQ SCEERNLREN

2101 GYEAEWRYNS CAPACOVTCO HPEPLACPVO CVEGCHAHCP PGKILDELLQ

2151 TCVDPEDCPV CEVAGRRFAS GKKVTLNPSD PEHCQICHCD

VVNLTCEACO 2201 EPISGTSESA TPESGPGSEP ATSGSETPGT SESATPESGP GSEPATSGSE

2251 TPGTSESATP ESGPGTSTEP SEGSAPGSPA GSPTSTEEGT SESATPESGP

2301 GSEPATSGSE TPGTSESATP ESGPGSPAGS PTSTEEGSPA GSPTSTEEGT

2351 STEPSEGSAP GTSESATPES GPGTSESATP ESGPGTSESA TPESGPGSEP

2401 ATSGSETPGS EPATSGSETP GSPAGSPTST EEGTSTEPSE GSAPGTSTEP

2451 SEGSAPGSEP ATSGSETPGT SESATPESGP GTSTEPSEGS APDSGGGGSG

2501 GGGSGGGGSG GGGSGGGGSL VPRGSGGDKT HTCPPCPAPE

LLGGPSVFLF

2551 PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE

VHNAKTKPRE

2601 EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALPAPIE

KTISKAKGQP

2651 REPQVYTLPP SRDELTKNQV SLTCLVKGFY PSDIAVEWES

NGQPENNYKT

2701 TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH

NHYTQKSLSL

2751 SPGK

(b) Cloning of pSYN-FVIII-168, 175, 172 and 174 (Figure 4A-4D)

[0352] pSYN-FVIII-168, 172, 174 and 175 are derivatives of pSYN-FVIII-161.

R1645A/R1648A mutations were introduced into pSYN-FVIII-161 to form pSYN-FVIII-168, which produces a SC-FVIII isoform, and an AE288 XTEN was directly fused into the C-terminus of FVIII-HC for further half- life extension. To construct pSYN-FVIII-175, the D'D3 codon sequence was remove form pSYN- FVIII-168 for evaluation of the effect of Fc and XTEN technology on FVIII half- life extension.

[0353] To construct pSYN-FVIII- 172, the AE288 XTEN fragment was directly fused into the C-terminus of FVIII-HC for further half-life extension, and the D'D3 codon sequence was removed from pSYN-FVIII-172 to form pSYN-FVIII- 174 for evaluation of the effect of Fc and XTEN technology on FVIII half-life extension.

(c) Cloning of pSYN-FVIII-170 (Figure 4E)

[0354] pS YN-FVIII- 170 was constructed to evaluate the effect of XTEN and

D'D3 fragment on FVIII half-life extension. The codon sequence VWF- D1D2D'D3 fragment and BDD-FVIII were introduced into the 5' and 3' end of expression casket, an AE288 XTEN codon sequence which followed by a 35 aa thrombin cleavable linker was used to connect the VWF and FVIII molecule. After intra cellular processing, the secreted protein comprises a polypeptide contains the D'D3 fragment of mature VWF molecule which is linked to the N- terminus of mature BDD-FVIII by an AE288 XTEN/35 aa thrombin cleavable linker.

pSYN-FVIII-170 protein sequence (SEP ID NO: 102)

1 SLSCRPPMVK LVCPADNLRA EGLECTKTCQ NYDLECMSMG CVSGCLCPPG

51 MVRHENRCVA LERCPCFHQG KEYAPGETVK IGCNTCVCRD RKWNCTDHVC

101 DATCSTIGMA HYLTFDGLKY LFPGECQYVL VQDYCGSNPG

TFRILVGNKG

151 CSHPSVKCKK RVTILVEGGE IELFDGEVNV KRPMKDETHF

EVVESGRYII

201 LLLGKALSVV WDRHLSISVV LKQTYQEKVC GLCGNFDGIQ

NNDLTSSNLQ

251 VEEDPVDFGN SWKVSSQCAD TRKVPLDSSP ATCHNNIMKQ

TMVDSSCRIL

301 TSDVFQDCNK LVDPEPYLDV CIYDTCSCES IGDCAAFCDT

IAAYAHVCAQ

351 HGKVVTWRTA TLCPQSCEER NLRENGYEAE WRYNSCAPAC

QVTCQHPEPL

401 ACPVQCVEGC HAHCPPGKIL DELLQTCVDP EDCPVCEVAG

RRFASGKKVT

451 LNPSDPEHCQ ICHCDVVNLT CEACQEPISG TSESATPESG

PGSEPATSGS

501 ETPGTSESAT PESGPGSEPA TSGSETPGTS ESATPESGPG

TSTEPSEGSA

551 PGSPAGSPTS TEEGTSESAT PESGPGSEPA TSGSETPGTS

ESATPESGPG

601 SPAGSPTSTE EGSPAGSPTS TEEGTSTEPS EGSAPGTSES

ATPESGPGTS

651 ESATPESGPG TSESATPESG PGSEPATSGS ETPGSEPATS

GSETPGSPAG

701 SPTSTEEGTS TEPSEGSAPG TSTEPSEGSA PGSEPATSGS

ETPGTSESAT

751 PESGPGTSTE PSEGSAPDSG GGGSGGGGSG GGGSGGGGSG

GGGSLVPRGS

801 GGASATRRYY LGAVELSWDY MQSDLGELPV DARFPPRVPK

SFPFNTSVVY

851 KKTLFVEFTD HLFNIAKPRP PWMGLLGPTI QAEVYDTVVI

TLKNMASHPV

901 SLHAVGVSYW KASEGAEYDD QTSQREKEDD KVFPGGSHTY

VWQVLKENGP

951 MASDPLCLTY SYLSHVDLVK DLNSGLIGAL LVCREGSLAK

EKTQTLHKFI 1001 LLFAVFDEGK SWHSETKNSL MQDRDAASAR AWPKMHTVNG YVNRSLPGLI

1051 GCHRKSVYWH VIGMGTTPEV HSIFLEGHTF LVRNHRQASL EISPITFLTA

1101 QTLLMDLGQF LLFCHISSHQ HDGMEAYVKV DSCPEEPQLR MKNNEEAEDY

1151 DDDLTDSEMD VVRFDDDNSP SFIQIRSVAK KHPKTWVHYI AAEEEDWDYA

1201 PLVLAPDDRS YKSQYLNNGP QRIGRKYKKV RFMAYTDETF KTREAIQHES

1251 GILGPLLYGE VGDTLLI IFK NQASRPYNIY PHGITDVRPL YSRRLPKGVK

1301 HLKDFPILPG EIFKYKWTVT VEDGPTKSDP RCLTRYYSSF VNMERDLASG

1351 LIGPLLICYK ESVDQRGNQI MSDKRNVILF SVFDENRSWY LTENIQRFLP

1401 NPAGVQLEDP EFQASNIMHS INGYVFDSLQ LSVCLHEVAY WYILSIGAQT

1451 DFLSVFFSGY TFKHKMVYED TLTLFPFSGE TVFMSMENPG LWILGCHNSD

1501 FRNRGMTALL KVSSCDKNTG DYYEDSYEDI SAYLLSKNNA IEPRSFSQNP

1551 PVLKRHQREI TRTTLQSDQE EIDYDDTISV EMKKEDFDIY DEDENQSPRS

1601 FQKKTRHYFI AAVERLWDYG MSSSPHVLRN RAQSGSVPQF KKVVFQEFTD

1651 GSFTQPLYRG ELNEHLGLLG PYIRAEVEDN IMVTFRNQAS RPYSFYSSLI

1701 SYEEDQRQGA EPRKNFVKPN ETKTYFWKVQ HHMAPTKDEF DCKAWAYFSD

1751 VDLEKDVHSG LIGPLLVCHT NTLNPAHGRQ VTVQEFALFF TIFDETKSWY

1801 FTENMERNCR APCNIQMEDP TFKENYRFHA INGYIMDTLP GLVMAQDQRI

1851 RWYLLSMGSN ENIHS IHFSG HVFTVRKKEE YKMALYNLYP GVFETVEMLP

1.901 SKAGIWRVEC LIGEHLHAGM STLFLVYSNK CQTPLGMASG HIRDFQITAS

1951 GQYGQWAPKL ARLHYSGSIN AWSTKEPFSW IKVDLLAPMI IHGIKTQGAR

2001 QKFSSLYISQ FIIMYSLDGK KWQTYRGNST GTLMVFFGNV DSSGIKHNIF

2051 NPPIIARYIR LHPTHYS IRS TLRMELMGCD LNSCSMPLGM ESKAISDAQI

2101 TASSYFTNMF ATWSPSKARL HLQGRSNAWR PQVNNPKEWL QVDFQKTMKV

2151 TGVTTQGVKS LLTSMYVKEF LISSSQDGHQ WTLFFQNGKV KVFQGNQDSF

2201 TPVVNSLDPP LLTRYLRIHP QSWVHQIALR MEVLGCEAQD LY Example 4: Hydrodynamic injection of XTEN containing FVIIIF/VWF constructs in FVIII and VWF deficient mice

[0355] The XTEN containing DNA constructs in Figures 3 and 4 have combined

2-3 half-life extension elements together. To evaluate their FVIII half-life extension potential, a selective group of DNA constructs in figure 3 and figure 4 were introduced into FVIII/VWF double knockout (DKO) mice by Hydrodynamic injection (HDI) at lOOug/mouse dose. Blood samples were then collected by retro orbital blood collection at 24hr post HDI. The post HDI plasma FVIII activity was analyzed by FVIII chromogenic assay, and results were listed in Table 17 and Figure 5. Compared to wild type BDD-FVIII, all XTEN containing DNA constructs yield significantly higher FVIII plasma activity at 24hr post HDI, indicating the corresponding molecules had significant longer circulating protein half-life than BDD-FVIII. The application of the combination of those half-life extending elements was further evaluated in Hemophilic animals.

TABLE 17: FVIII plasma activity 24hr post HDI in FVIII/VWF DKO mice

Hydrodynamic injection:

[0356] Hydrodynamic Injection is an efficient and safe non-viral gene delivery method to the liver in small animals, such as mice and rats. It was originally described as a rapid injection of a naked plasmid DNA/saline solution free of endotoxin at a tenth volume of the animal's body weight in about 5-7 seconds. The naked plasmid DNA contains the gene of interest and the liver produced in a tenth volume of the animal^'s body weight. The targeted protein is produced in the liver from the injected DNA and can be detected within 24 hours post-injection. Plasma samples were then collected to study the therapeutic property of the expressed protein.

[0357] For all the hydrodynamic injections that were performed herein, 2 ml of plasmid DNA in 0.9% sterile saline solution was delivered via intravenous tail vein injection within about 4-7 seconds to mice weighing 20-35 grams. The mice were closely monitored for the first couple of hours until the normal activity resumed. After the blood samples were collected via retro orbital blood collection, plasma samples were then obtained and stored at -80 °C for further analysis.

Example 5: Plasmid construction of co-transfection system for FVIIIFc-VWF Heterodimer contain XTEN insertions (Figure 6)

To increase the protein production yield, two co-transfection systems were generated for protein production, which contains three DNA constructs. The first DNA construct encoded a FVIII-Fc fusion protein in which a AE288 XTEN fragment was directly fuse to the C-terminus of the FVIII heavy chain and followed by either a wild type FVIII light chain fragment (pSYN-FVIII-173, Figure 6B) or a FVIII light chain fragment with R1645A/R1648A mutations (pSYN-FVIII-169, Figure 6A), the FVIII light chain was then directly fused to a single Fc fragment. The second DNA construct is pSYN-VWF-031 which encoding a D'D3-Fc fusion protein (Example 1). HEK293F cells were transfected with the two plasmid along with a third plasmid (PC5) at 80: 15:5 ratio. The synthesized proteins were secreted as FVIII (XTEN) Fc/D'D3Fc heterodimer and D'D3Fc dimer and the FVIII (XTEN) Fc/D'D3Fc heterodimer was separated from the D'D3Fc dimer by protein purification.

pSYN-FVIII-169 mature Protein sequence (SEP ID NO: 103):

1 ATRRYYLGAV ELSWDYMQSD LGELPVDARF PPRVPKSFPF

NTSVVYKKTL

51 FVEFTDHLFN IAKPRPPWMG LLGPTIQAEV YDTVVITLKN

MASHPVSLHA

101 VGVSYWKASE GAEYDDQTSQ REKEDDKVFP GGSHTYVWQV

LKENGPMASD

151 PLCLTYSYLS HVDLVKDLNS GLIGALLVCR EGSLAKEKTQ

TLHKFILLFA

201 VFDEGKSWHS ETKNSLMQDR DAASARAWPK MHTVNGYVNR

SLPGLIGCHR

251 KSVYWHVIGM GTTPEVHSIF LEGHTFLVRN HRQASLEISP

ITFLTAQTLL

301 MDLGQFLLFC HISSHQHDGM EAYVKVDSCP EEPQLRMKNN

EEAEDYDDDL

351 TDSEMDVVRF DDDNSPSFIQ IRSVAKKHPK TWVHYIAAEE

EDWDYAPLVL

401 APDDRSYKSQ YLNNGPQRIG RKYKKVRFMA YTDETFKTRE

AIQHESGILG

451 PLLYGEVGDT LLI IFKNQAS RPYNIYPHGI TDVRPLYSRR

LPKGVKHLKD 501 FPILPGEIFK YKWTVTVEDG PTKSDPRCLT RYYSSFVNME

RDLASGLIGP

551 LLICYKESVD QRGNQIMSDK RNVILFSVFD ENRSWYLTEN

IQRFLPNPAG

601 VQLEDPEFQA SNIMHS INGY VFDSLQLSVC LHEVAYWYIL

SIGAQTDFLS

651 VFFSGYTFKH KMVYEDTLTL FPFSGETVFM SMENPGLWIL

GCHNSDFRNR

701 GMTALLKVSS CDKNTGDYYE DSYEDI SAYL LSKNNAIEPR

SFSQNGAPGT

751 SESATPESGP GSEPATSGSE TPGTSESATP ESGPGSEPAT

SGSETPGTSE

801 SATPESGPGT STEPSEGSAP GSPAGSPTST EEGTSESATP

ESGPGSEPAT

851 SGSETPGTSE SATPESGPGS PAGSPTSTEE GSPAGSPTST

EEGTSTEPSE

901 GSAPGTSESA TPESGPGTSE SATPESGPGT SESATPESGP

GSEPATSGSE

951 TPGSEPATSG SETPGSPAGS PTSTEEGTST EPSEGSAPGT

STEPSEGSAP

1001 GSEPATSGSE TPGTSESATP ESGPGTSTEP SEGSAPASSP

PVLKRHQAEI

1051 TRTTLQSDQE EIDYDDTISV EMKKEDFDIY DEDENQSPRS

FQKKTRHYFI

1101 AAVERLWDYG MSSSPHVLRN RAQSGSVPQF KKVVFQEFTD

GSFTQPLYRG

1151 ELNEHLGLLG PYIRAEVEDN IMVTFRNQAS RPYSFYSSLI

SYEEDQRQGA

1201 EPRKNFVKPN ETKTYFWKVQ HHMAPTKDEF DCKAWAYFSD

VDLEKDVHSG

1251 LIGPLLVCHT NTLNPAHGRQ VTVQEFALFF TIFDETKSWY

FTENMERNCR

1301 APCNIQMEDP TFKENYRFHA INGYIMDTLP GLVMAQDQRI

RWYLLSMGSN

1351 ENIHS IHFSG HVFTVRKKEE YKMALYNLYP GVFETVEMLP

SKAGIWRVEC

1.401 LIGEHLHAGM STLFLVYSNK CQTPLGMASG HIRDFQITAS

GQYGQWAPKL

1451 ARLHYSGSIN AWSTKEPFSW IKVDLLAPMI IHGIKTQGAR

QKFSSLYISQ

1501 FIIMYSLDGK KWQTYRGNST GTLMVFFGNV DSSGIKHNIF

NPPIIARYIR

1551 LHPTHYS IRS TLRMELMGCD LNSCSMPLGM ESKAISDAQI

TASSYFTNMF

1601 ATWSPSKARL HLQGRSNAWR PQVNNPKEWL QVDFQKTMKV

TGVTTQGVKS

1651 LLTSMYVKEF LISSSQDGHQ WTLFFQNGKV KVFQGNQDSF

TPVVNSLDPP

1.701 LLTRYLRIHP QSWVHQIALR MEVLGCEAQD LYDKTHTCPP

CPAPELLGGP 1751 SVFLFPPKPK DTLMI SRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK

1801 TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK

1851 AKGQPREPQV YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE

1901 NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM

HEALHNHYTQ

1951 KSLSLSPGK

pSYN-FVIII-173 mature Protein sequencing (SEP ID NO: 104):

1 ATRRYYLGAV ELSWDYMQSD LGELPVDARF PPRVPKSFPF NTSVVYKKTL

51 FVEFTDHLFN IAKPRPPWMG LLGPTIQAEV YDTVVITLKN MASHPVSLHA

101 VGVSYWKASE GAEYDDQTSQ REKEDDKVFP GGSHTYVWQV LKENGPMASD

151 PLCLTYSYLS HVDLVKDLNS GLIGALLVCR EGSLAKEKTQ TLHKFILLFA

201 VFDEGKSWHS ETKNSLMQDR DAASARAWPK MHTVNGYVNR SLPGLIGCHR

251 KSVYWHVIGM GTTPEVHSIF LEGHTFLVRN HRQASLEISP ITFLTAQTLL

301 MDLGQFLLFC HISSHQHDGM EAYVKVDSCP EEPQLRMKNN EEAEDYDDDL

351 TDSEMDVVRF DDDNSPSFIQ IRSVAKKHPK TWVHYIAAEE EDWDYAPLVL

401 APDDRSYKSQ YLNNGPQRIG RKYKKVRFMA YTDETFKTRE AIQHESGILG

451 PLLYGEVGDT LLI IFKNQAS RPYNIYPHGI TDVRPLYSRR LPKGVKHLKD

501 FPILPGEIFK YKWTVTVEDG PTKSDPRCLT RYYSSFVNME RDLASGLIGP

551 LLICYKESVD QRGNQIMSDK RNVILFSVFD ENRSWYLTEN IQRFLPNPAG

601 VQLEDPEFQA SNIMHS INGY VFDSLQLSVC LHEVAYWYIL SIGAQTDFLS

651 VFFSGYTFKH KMVYEDTLTL FPFSGETVFM SMENPGLWIL GCHNSDFRNR

701 GMTALLKVSS CDKNTGDYYE DSYEDI SAYL LSKNNAIEPR SFSQNGAPGT

751 SESATPESGP GSEPATSGSE TPGTSESATP ESGPGSEPAT SGSETPGTSE

801 SATPESGPGT STEPSEGSAP GSPAGSPTST EEGTSESATP ESGPGSEPAT

851 SGSETPGTSE SATPESGPGS PAGSPTSTEE GSPAGSPTST EEGTSTEPSE

901 GSAPGTSESA TPESGPGTSE SATPESGPGT SESATPESGP GSEPATSGSE

951 TPGSEPATSG SETPGSPAGS PTSTEEGTST EPSEGSAPGT STEPSEGSAP 1001 GSEPATSGSE TPGTSESATP ESGPGTSTEP SEGSAPASSP

PVLKRHQREI

1051 TRTTLQSDQE EIDYDDTISV EMKKEDFDIY DEDENQSPRS

FQKKTRHYFI

1101 AAVERLWDYG MSSSPHVLRN RAQSGSVPQF KKVVFQEFTD

GSFTQPLYRG

1151 ELNEHLGLLG PYIRAEVEDN IMVTFRNQAS RPYSFYSSLI

SYEEDQRQGA

1201 EPRKNFVKPN ETKTYFWKVQ HHMAPTKDEF DCKAWAYFSD

VDLEKDVHSG

1251 LIGPLLVCHT NTLNPAHGRQ VTVQEFALFF TIFDETKSWY

FTENMERNCR

1301 APCNIQMEDP TFKENYRFHA INGYIMDTLP GLVMAQDQRI

RWYLLSMGSN

1351 ENIHS IHFSG HVFTVRKKEE YKMALYNLYP GVFETVEMLP

SKAGIWRVEC

1401 LIGEHLHAGM STLFLVYSNK CQTPLGMASG HIRDFQITAS

GQYGQWAPKL

1451 ARLHYSGSIN AWSTKEPFSW IKVDLLAPMI IHGIKTQGAR

QKFSSLYISQ

1501 FIIMYSLDGK KWQTYRGNST GTLMVFFGNV DSSGIKHNIF

NPPIIARYIR

1551 LHPTHYS IRS TLRMELMGCD LNSCSMPLGM ESKAISDAQI

TASSYFTNMF

1601 ATWSPSKARL HLQGRSNAWR PQVNNPKEWL QVDFQKTMKV

TGVTTQGVKS

1651 LLTSMYVKEF LISSSQDGHQ WTLFFQNGKV KVFQGNQDSF

TPVVNSLDPP

1701 LLTRYLRIHP QSWVHQIALR MEVLGCEAQD LYDKTHTCPP

CPAPELLGGP

1751 SVFLFPPKPK DTLMI SRTPE VTCVVVDVSH EDPEVKFNWY

VDGVEVHNAK

1801 TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL

PAPIEKTISK

851 AKGQPREPQV YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA

VEWESNGQPE

1.901 NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM

HEALHNHYTQ

1951 KSLSLSPGK

Example 6. Protein purification for FVIII- 169/VWF-031 and FVIII- 173/VWF- 031

A tangential flow filtration (TFF) step was used to buffer exchange the clarified conditioned media. The FVIII- 169/VWF-031 or FVIII- 173/VWF-031 heterodimer was then purified using a two-step chromatography process. A weak anion exchange resin was used, followed by affinity chromatography. The final purified product had acceptable purity by SEC-HPLC. The specific activity was compatible to B-domain deleted FVIII, as measured by FVIII chromogenic assay and A280 concentration. Purity and the presence of each moiety of this molecule were confirmed by SDS-PAGE and western blotting.

Example 7. Evaluation the VWF binding ability of FVIII- 169/VWF-031 by Octet assay

[0360] The VWF binding ability of FVIII- 169/VWF-031 was obtained by Bio-

Layer Interferometry (BLI) based measurements (Octet assay) at 25 °C with a ForteBio Octet 384 instrument, using Tris binding buffer (50 mM Tris, pH 7.2, 150 mM NaCl, 5 mM CaCl₂). The Octet assay for determining FVIII binding was based on the hydrophobic immobilization of Human von Willebrand Factor (Haematologic Technologies Catalog No. HCVWF-0191) onto the APS

Biosensor, then followed by the binding of 1.0% Bovine Serum Albumin (Jackson ImmunoResearch Catalog No. 001-000-161). Briefly, hvWF (20 μg/mL) was diluted in Tris buffer and loaded across APS Biosensors for 600 sec, yielding approximately 3.0 - 3.5 nm binding on the reaction probes. Control APS probes were loaded with 1.0% BSA in the absence of hvWF for reference subtraction. After loading, all probes were incubated in Tris buffer for 300 sec to establish a new baseline. Subsequently, biosensor probes were incubated in solutions of FVIII-XTEN 169 or FVIIIFc Drug Substance (0, 0.6, 2, 6, 20, 60, 200, 600 IU/mL) for 5 min at room temperature, followed by a 5 min dissociation step. Using the Octet data analysis software, the binding response (nm) was derived from the subtracted data (Reaction probe minus Reference probe). No binding to immobilized VWF was detected for FVIII- 169/VWF-031 (Figure 7), indicating a complete shielding of FVIII from full length VWF molecule by the D'D3 fragment.

Example 8. FVIII- 169/VWF-031 PK in HemA and FVIII/VWF DKO mice

[0361] The PK profile of FVIII- 169/VWF-031 was tested in HemA and

FVIII/VWF DKO mice to evaluate the ability of the D'D3 fragment to shield the FVIII moiety from the endogenous VWF. HemA or FVIII/VWF DKO mice were treated with a single intravenous dose of FVIII- 169/VWF-031 at 200 IU/kg, plasma samples were then collected at 5min, 8hr, 24hr, 48hr and 72 hours post dosing. The FVIII activity of plasma sample was tested by FVIII chromogenic assay, and half-life of FVIII-169/VWF-031 was calculated using WinNonlin program.

[0362] Complete inhibition of the constructs' binding to immobilized VWF was demonstrated by biolayer interferometry (Figure 7) for FVIII-169/VWF-031. This indicates the D'D3 fragment in the molecule had successfully blocked the FVIII binding to native VWF molecules, therefor similar half-life of FVIII- 169/VWF- 031 was predicted in the two different mouse strains. As shown in Figure 8A and Table 18, as expected, FVIII- 169 /VWF-031 had similar PK profile in both HemA and FVIII/VWF DKO mice, which has demonstrated that the half-life of

FVIIIFc/VWF heterodimer is independent from the half-life of endogenous VWF. The separation of the FVIIIFc/VWF heterodimer half-life from the endogenous VWF half-life , eliminated the FVIII extension ceiling and opened the possibility of further extending FVIII half-life beyond the 2 fold half-life limit imposed by endogenous VWF.

TABLE 18: FVIII-169/VWF-031 PK in HemA and FVIII/VWF DKO mice

[0363] The FVIII protecting ability of the XTEN insertion and D'D3 fragment was evaluated by comparing the half-life of FVIII- 169/VWF-031 with FVIII- 169/Fc and FVIIIFc in FVIII/VWF DKO mice. After a single IV administration, blood samples were collected at 5min, 8hr, 24hr, 48hr and 72hr for FVIII- 169/VWF- 031, 5min, 8hr, 24hr, 32hr, 48hr for FVIII- 169/Fc and at 5min, 1, 2, 4, 6 and 8hrs for FVIIIFc. The FVIII activity of plasma sample was tested by FVIII

chromogenic assay, and half-life of FVIII-155/VWF-031 was calculated using WinNonlin program.

[0364] The study results were summarized in Figure 8B and Table 19, rFVIIIFc has a 1.6hr half- life in DKO mice due to the loss of VWF protection. When an XTEN insertion was introduced into the FVIIIFc molecule, the resulting FVIII- 169/Fc molecule has a 7hr half-life, a 4 fold half-life extension by the XTEN insertion. Finally, when D'D3 fragment was incorporated into the molecule to form FVIII- 169/VWF-031, a 17hr half-life was observed, another 2.5 fold further increase by the D'D3 fragment. In addition of the half-life improvement, improved Mean residency time (MRT), Clearance (CI) and AUC were also observed as shown in Table 19.

[0365] FVIII- 169/VWF-031 has achieved 17- 18hr t_m in both HemA and

FVIII/VWF DKO mice, which is the upper limit of the tm extension ceiling that imposed by VWF clearance. More t extension elements can be further incorporated into this molecule, such as a second XTEN insertion within FVIII. The synergistic effect of D'D3 fragment and XTEN insertions provided the possibility of the complete protection for FVIII from its clearance pathway, a final breakthrough of the 2 fold FVIII t extension limit might be achieved by the FVIIIFc/XTEN/VWF variants.

TABLE 19. FVIII-169/VWF-031 PK in FVIII/VWF DKO mice

Example 9: FVIII-XTEN variants cell media concentrate PK in D'D3 expressing FVIII/VWF DKO mice

[0366] The ability of D'D3 fragment to extend the t_m of FVII-XTEN was

evaluated in the D'D3 expressing FVIII/VWF DKO mouse model (described in example 2). In this study, instead of using VWF-025 to introduce the D'D3 dimer into the circulation, VWF-029 construct was used to introduce the D'D3 monomer into the circulation. To prepare FVIII-XTEN variants protein, a small scale (50- 100 mL) transient transfection culture media was prepared, at day 4 post transfection, cell culture was harvested and concentrated to reach 10-20 IU/mL of FVIII activity range which is suitable for PK study. The concentrated cell media were then used for standard PK study in FVIII/VWF DKO mice with or without D'D3 in the circulation.

[0367] Total of 6 FVIII-XTEN variants that contains 1-3 XTEN insertions were tested in the system, their t ₂ were summarized in Table 20 and data from representative variants were plotted in Figure 9A.

[0368] Longer half-life was observed for all the FVIII-XTEN variants with the presents of D'D3 fragment in the circulation (Table 20), which demonstrated the D'D3 protection for FVIII-XTEN from its clearance pathways. Furthermore, when compared to its 14hr half-life in HemA mice, LSD0055.021 has a 20.4hr t ₂ in D'D3 expressing DKO mice (Figure 9B, Table 20), indicates the final breakthrough of the 2 fold half-life extension ceiling for FVIII molecules. By further modify the FVIII(XTEN)/VWF molecule, we could potentially achieve even longer FVIII t , and provide HemA patients a FVIII protein that only requires once weekly or less frequent dosing regimen.

TABLE 20. FVIII-XTEN t_1/2 in D'D3 expressing FVIII/VWF DKO mice

*B indicates an XTEN sequence (e.g., 144) is inserted immediately downstream of amino acid residue 745 corresponding to mature FVIII sequence.

Example 10: Stability of VWF- and XTEN-containing FVIII variants in

FVIII/VWF double knockout (DKO) plasma

[0369] Plasma stability of rFVIIIFc protein variants was tested in FVIII/VWF double knockout (DKO) mouse plasma. For the stability assay, HEK293 cells were co-transfected with plasmids directing the expression of rFVIIIFc or FVIII - 169 (rFVIIIFc with 288 AE XTEN inserted at the B-domain junction) and plasmids directing the expression of either IgG-Fc or VWF-031 (VWF D'D3 region fused to IgG-Fc). At day four post-transfection, cell culture media was harvested and concentrated to 30 IU/mL based on FVIII chromogenic activity. Concentrated cell culture medium was then added into DKO mouse plasma to yield a FVIII activity of 5 IU/mL and incubated at 37°C. Aliquots were collected at different time points for activity measurement by chromogenic assay. Activity at each time point was measured in duplicate, and the average activity was plotted as a function of time. The activity of FVIIIFc, a dual chain (dc) FVIII molecule in which heavy and light chains are held together by non-covalent interaction, decreases with time in DKO mouse plasma (Figure 10). The activity of FVIII - 169:Fc, which contains a 288 AE XTEN insertion at the B-domain junction, decays at a reduced rate relative to rFVIIIFc, indicating that enhanced stability is conferred by the XTEN insertion. Given that VWF has been proposed to enhance the stability of FVIII in vivo, we evaluated the plasma stability of FVIII- 169:VWF-031. This heterodimeric molecule, in which the FVIII element and the VWF D'D3 element are fused to respective hemi-domains of Fc, exhibited additional plasma stability relative to FVIII- 169 :Fc, indicating that the VWF D'D3 domain and XTEN have a synergistic effect on the plasma stability of rFVIIIFc. Example 11 : The effect on FVIII half-life of Fc fusion, XTEN insertion and the D^*D3 fragment of VWF.

[0370] To assess the effect of Fc fusion, XTEN insertion and D'D3 fragment of

VWF on the half-life of FVIII, the pharmacokinetic properties of B domain deleted recombinant FVIII (rBDD-FVIII), rFVIIIFc, FVIII- 169 :Fc and FVIII- 169:VWF-031 were evaluated in FVIII/VWF double knockout (DKO) mice.

[0371] DKO mice were treated with a single intra venous administration of 200

IU/kg of FVIII proteins, and plasma samples were collected at designated time points as indicated in Figure 11. FVIII activity of the plasma samples were analyzed by FVIII chromogenic assay and half-life was calculated using the WinNonlin-Phoenix program. The pharmacokinetic parameters of the tested molecules are listed in Table 21. The time regression curve of plasma FVIII activity for each FVIII variants were plotted in Figure 11.

[0372] Unmodified BDD-FVIII had a half-life of 0.23 hr in DKO mice, the

FVIIIFc fusion protein has an extended half-life of 1.66 hr in DKO mice due to the recycling of FVIIIFc protein through the Fc:FcRn interaction. When a 288 residue of AEXTEN polypeptide was incorporated into the B domain region of FVIII within the FVIIIFc molecule, the half-life of the resulting FVIII169/Fc protein was further extended to 7.41 hr in DKO mice. Finally, with the addition of the D*D3 domain of VWF, the half-life of FVIII 169/VWF031 heterodimer has reached 17.9 hr in DKO mice (Figure 1 1 , Table 21). In addition of the half-life, all of the other PK parameters also improved proportionally with the addition of each element (Table 21). FVIII can tolerate multiple half-life extension elements, and this synergistic effect of the three elements on FVIII half-life extension, enabled the further improvement of the half-life of FVIII-XTEN VWF heterodimers. Table 21: PK parameters of FVIII variants

Example 12: Pharmacokinetic properties of different FVIII-XTEN VWF heterodimers

[0373] To evaluate the combined effect of the VWF-DO3 fragment and XTEN insertions on the FVIII half-life, the pharmacokinetic properties of FVIII-XTEN- Fc:VWF-Fc heterodimers were tested in HemA mice and compared to those of the single chain isoform of BDD-FVIII (scBDD-FVIII) and FVIII- 169 :VWF-031 (example 10). Seven new FVIII -XTEN-Fc constructs were generated (protein sequences were listed in Table 24). Schematic diagrams of those constructs are shown in Figure 14A-H. FVIII- 195 and FVIII- 199, respectively, are the FVIII dual chain and single chain isoforms that each contains two XTEN insertions at positions 1900 and 1656. FVIII- 196 and FVIII-201 , respectively, are the FVIII dual chain and single chain isoforms that each contains three XTEN insertions at positions 26, 1656 and 1900. FVIII-203, -204 and -205 are sc-FVIIIFc molecules with two XTEN insertions at the B domain junction and at positions 1900, 403 or 18, respectively. Each FVIII -XTEN-Fc construct was co-expressed with VWF- 031 in HEK293 cells to produce FVIII-XTEN-Fc/VWF heterodimeric proteins. At day four post-transfection, cell culture medium was harvested and either concentrated to 20 IU/mL based on FVIII chromogenic activity (FVIII- 195 :VWF- 031, FVIII- 196 :VWF-031, FVIII- 199 :VWF-031, FVIII-203 : VWF-031 and FVIII- 204:VWF-031) or purified (scBDD-FVIII, FVIII- 169: VWF-031, FVIII- 201 : VWF-031 and FVIII-205 : VWF-031 ). Having demonstrated the complete intra-molecular shielding of FVIII molecule from the endogenous VWF by the D^*D3 fragment in the FVIII-XTEN-Fc:VWF-Fc heterodimer (FVIII- 169 : VWF- 031, Example 5), HemA mice was chosen for the PK evaluations. Purified protein or concentrated cell culture medium was administered to 8-12 week-old HemA mice by intravenous administration at a dose of 200 IU/10 mL/kg. Plasma samples were collected at 5 min, 8 hr, 16 hr, 24 hr, 32 hr, 48 hr, 72 hr and 96 hr post-dosing. FVIII activity of the plasma samples were analyzed by FVIII chromogenic assay and half-life was calculated using the WinNonlin-Phoenix program. The pharmacokinetic parameters of the tested molecules are listed in Table 22. The plasma FVIII activities at selected time points for FVIII-XTEN- Fc/VWF-Fc variants were plotted in Figures 12A-C.

When XTEN was inserted into positions 1900 and 1656 (FVIII-195,

FVIII- 199), moderate improvement in half-life was observed for the scFVIII isoform (FVIII- 199: VWF-031) compared to FVIII- 169: VWF-031. However, the dcFVIII isoform exhibited a shorter half-life than did FVIII- 169: VWF-031, indicating that the single chain isoform might be significantly more stable than the corresponding dual chain isoform (Table 22 and Figure 12A). When a third XTEN insertion was incorporated into FVIII- 199 at position 26, the half-life of the resulting molecule FVIII-201 : VWF-031 had reached 24.6 hr, which represents greater than a threefold half-life improvement relative to scBDD-FVIII (Table 22 and Figure 12C). We have also tested the half-life extension effect of the second XTEN insertion at position 403 (A2 domain), 1900 (A3 domain) and 18 (A 1 domain) each in combination with the B domain XTEN insertion. While the addition of the A2 or A3 XTEN insertion did not confer an additional half-life benefit (Table 22, Figure 12b), the addition of the Al insertion further extended the half-life of the FVIII-XTEN-Fc:VWF-Fc heterodimer to 29.4 hr (Table 22, Figure 12C), which is greater than threefold longer than that of scBDD-FVIII. [0375] When XTENs were incorporated into the FVIIIFc/VWF heterodimer construct, degree of half-life improvement of the resulting molecules was variable, and no obvious correlation was observed between half-lives and either the site or number of XTEN insertion, suggesting that the half-life of the FVIII-XTEN- Fc/VWF heterodimer is determined by the integrity of the whole molecule rather than by the number or placement of XTEN insertions.

[0376] The 24.6 hr and 29.4 hr half-lives observed for FVIII-XTEN-Fc:VWF-Fc heterodimers clearly exceeded the 1.6- to 2-fold limitation on FVIII half-life extension. If this finding translates for HemA patients, it will allow once-weekly or less frequent dosing for FVIII prophylaxis.

Table 22: PK parameters of FVIII-XTEN-Fc/VWF-Fc heterodimers

[0377] In addition to incorporating XTEN into the FVIII molecule, we also

evaluated the potential half-life extension benefit of incorporating XTEN as a linker between the D'D3 and Fc fragment. FVIII-155 (scFVIIIFc) was co- expressed with VWF-034 (VWF-Fc with AE 288 XTEN plus a 35 residue thrombin cleavable linker) in HEK293 cells. At day 4 post-transfection, cell culture medium was harvested and concentrated to 20 IU/mL based on FVIII activity assay. FVIII/VWF DKO mice were dosed with concentrated cell culture media at 200 IU/10 mL/kg with a single intravenous injection. Plasma samples were collected at 5 min, 8 hr, 24 hr, 48 hr, 72 hr and 96 hr post-dosing. The FVIII activity of plasma samples was analyzed by FVIII chromogenic assay, and the regression curve of plasma FVIII activity as a function of time was plotted (Figure 13). FVIII- 155 /VWF-034 exhibited the same improvement in half-life as FVIII- 169/VWF-031, which has AE 288 XTEN inserted into the B domain junction of FVIII, as illustrated by the over lapping regression curves for the two molecules (Figure 13). The demonstration that XTEN insertion into the VWF-Fc polypeptide confers half-life improvement of a magnitude similar to that conferred by XTEN insertion at the B domain junction of the FVIII polypeptide suggests that further half-life improvement may be possible in a heterodimeric molecule in which intra-molecular XTEN insertion in the FVIII polypeptide is combined with inter-domain XTEN insertion between the VWF and Fc elements of the VWF-Fc polypeptide.

Example 13 A: Pharmacokinetic properties of additional FVIII -XTEN VWF heterodimers

In addition to the FVIII-XTEN VWF heterodimers that were listed in

Table 22, FVIII-XTEN VWF heterodimers containing different composition of

XTEN insertions, single chain and dual chain version of FVIII (Table 23 A) are either tested or will be tested in HemA for their pharmacokinetic properties.

Various FVIII constructs (Table 23B) and VWF constructs (Table 23C) are also disclosed below. HemA mice will be treated with a single dose of intravenous administration of the heterodimer proteins at 200 IU/10 mL/kg. Plasma samples will then be collected at 5 min, 24, 48, 72, 96 and 120 hrs post-dosing. FVIII activity of the plasma samples will be analyzed by FVIII chromogenic assay and half-life will be calculated using the WinNonlin-Phoenix program. The protein sequences of the listed heterodimers were listed in Table 25.

Table 23A. Plausible FVIII-XTEN-Fc: VWF-Fc heterodimer combinations for PK and activity improvement.

pSYN FVIII t_1/216.7hr ti/231.1 hr

- - 169 ** HemA mice HemA mice

pSYN FVIII t_1/215.2hr t_1/228.9hr

- To be tested 173 ** DKO mice HemA mice

pSYN FVIII ti_/229.4hr ti_/232.4hr ti_/229.7hr 205 HemA mice HemA mice HemA mice pSYN FVIII ti_/224.5 hr ti_/227.4hr

- 266 HemA mice HemA mice

pSYN FVII - ti_/223.0hr ti_/225.7hr

267 HemA mice HemA mice pSYN FVIII

268 To be tested To be tested To be tested

Dual chain

isoform of To be tested To be tested To be tested pSYN FVIII

268

** Length of X^r ΓΕΝ can be changed to 72, 144, 288, 324, 333, 576, or 864.

Table 23B. FVIII Constructs:

269

pSYN FVIII Single chain FVIIIFc with 42 AE XTEN at amino acid 18

271

pSYN FVIII Single chain FVIII with 144 AE XTEN at amino acid 18 and 288 272 AE XTEN in B-domain (no Fc)

Table 23C. VWF Constructs:

pSYN VWF031 VWF-D 1 D2D'D3 - 48aa long thrombin cleavable GS linker-Fc with

C1099A/C1142A

pSYN VWF034 VWF-D 1 D2D'D3 - 288AE XTEN +35aa long thrombin cleavable

GS linker-Fc with C1099A/C1142 A

pSYN VWF035 VWF-D 1 D2D'D3 - 72aa long thrombin cleavable GS linker-Fc with

C1099A/C1142A

pSYN VWF036 VWF-D 1 D2D'D3 - 98aa long thrombin cleavable GS linker-Fc with

C1099A/C1142A

pSYN VWF041 VWF-D 1D2D'D3 with 288 AE XTEN in D3 and 48aa long

thrombin cleavable GS linker after D3-Fc with C1099A/C1142 A

Example 13B: Pharmacokinetic properties of additional FVIII-XTEN VWF heterodimers.

[0379] FVIII-XTEN VWF heterodimers were tested in HemA mice for their pharmacokinetic properties. The heterodimers tested are FVIII 169/VWF034, FVIII205/VWF034, FVIII205/VWF036 and FVIII266/VWF031. HemA mice were administered with a single intravenous dose of various heterodimer proteins at 200 IU/10 mL/kg. Plasma samples were collected at 5 min, 24, 48, 72, 96 and 120 hrs post-dosing. FVIII activity of the plasma samples were analyzed by FVIII chromogenic assay, and half-lives were calculated using the WinNonlin-Phoenix program. The PK results are shown below in Table 24.

Table 24. Additional FVIII-XTEN_VWF - PK in HemA Mice

031 pSYNFVIII 010 nucleotide sequence-(Dual chain FVIIIFc) (SEP ID NO:

125)

1 ATGCAAATAG AGCTCTCCAC CTGCTTCTTT CTGTGCCTTT TGCGATTCTG

51 CTTTAGTGCC ACCAGAAGAT ACTACCTGGG TGCAGTGGAA CTGTCATGGG

101 ACTATATGCA AAGTGATCTC GGTGAGCTGC CTGTGGACGC

AAGATTTCCT

151 CCTAGAGTGC CAAAATCTTT TCCATTCAAC ACCTCAGTCG

TGTACAAAAA

201 GACTCTGTTT GTAGAATTCA CGGATCACCT TTTCAACATC

GCTAAGCCAA

251 GGCCACCCTG GATGGGTCTG CTAGGTCCTA CCATCCAGGC

TGAGGTTTAT

301 GATACAGTGG TCATTACACT TAAGAACATG GCTTCCCATC

CTGTCAGTCT

351 TCATGCTGTT GGTGTATCCT ACTGGAAAGC TTCTGAGGGA

GCTGAATATG

401 A GA CAGAC CAGTCAAAGG GAGAAAGAAG ATGATAAAGT

CTTCCCTGGT

451 GGAAGCCATA CATATGTCTG GCAGGTCCTG AAAGAGAATG

GTCCAATGGC

501 CTCTGACCCA CTGTGCCTTA CCTACTCATA TCTTTCTCAT

GTGGACCTGG

551 TAAAAGACTT GAATTCAGGC CTCATTGGAG CCCTACTAGT

ATGTAGAGAA

601 GGGAGTCTGG CCAAGGAAAA GACACAGACC TTGCACAAAT

TTATACTACT

651 TTTTGCTGTA TTTGATGAAG GGAAAAGTTG GCACTCAGAA

ACAAAGAACT

701 CCTTGATGCA GGATAGGGAT GCTGCATCTG CTCGGGCCTG

GCCTAAAATG

751 CACACAGTCA ATGGTTATGT AAACAGGTCT CTGCCAGGTC

TGATTGGATG

801 CCACAGGAAA TCAGTCTATT GGCATGTGAT TGGAATGGGC

ACCACTCCTG

851 AAGTGCACTC AATATTCCTC GAAGGTCACA CATTTCTTGT

GAGGAACCAT

901 CGCCAGGCGT CCTTGGAAAT CTCGCCAATA ACTTTCCTTA

CTGCTCAAAC

951 ACTCTTGATG GACCTTGGAC AGTTTCTACT GTTTTGTCAT

ATCTCTTCCC

1001 ACCAACATGA TGGCATGGAA GCTTATGTCA AAGTAGACAG

CTGTCCAGAG

1051 GAACCCCAAC TACGAATGAA AAATAATGAA GAAGCGGAAG

ACTATGATGA

1101 TGATCTTACT GATTCTGAAA TGGATGTGGT CAGGTTTGAT

GATGACAACT 1151 CTCCTTCCTT TATCCAAATT CGCTCAGTTG CCAAGAAGCA TCCTAAAACT

1201 TGGGTACATT ACATTGCTGC TGAAGAGGAG GACTGGGACT ATGCTCCCTT

1251 AGTCCTCGCC CCCGATGACA GAAGTTATAA AAGTCAATAT GAACAATG

1301 GCCCTCAGCG GATTGGTAGG AAGTACAAAA AAGTCCGATT TATGGCATAC

1351 ACAGA GAAA CCTTTAAGAC TCGTGAAGCT ATTCAGCATG AATCAGGAAT

1401 CTTGGGACCT TTACTTTATG GGGAAGTTGG AGACACACTG TTGATTATAT

1451 TTAAGAATCA AGCAAGCAGA CCATATAACA TCTACCCTCA CGGAATCACT

1501 GATGTCCGTC CTTTGTATTC AAGGAGATTA CCAAAAGGTG TAAAACATTT

1551 GAAGGATTTT CCAATTCTGC CAGGAGAAAT ATTCAAATAT AAATGGACAG

1601 TGACTGTAGA AGATGGGCCA ACTAAATCAG ATCCTCGGTG CCTGACCCGC

1651 TATTACTCTA GTTTCGTTAA TATGGAGAGA GATCTAGCTT CAGGACTCAT

1701 TGGCCCTCTC CTCATCTGCT ACAAAGAATC TGTAGATCAA AGAGGAAACC

1751 AGATAATGTC AGACAAGAGG AATGTCATCC TGTTTTCTGT A GA GAG

1801 AACCGAAGCT GGTACCTCAC AGAGAATATA CAACGCTTTC TCCCCAATCC

1851 AGCTGGAGTG CAGCTTGAGG ATCCAGAGTT CCAAGCCTCC AACATCATGC

1901 ACAGCATCAA TGGCTATGTT TTTGATAGTT TGCAGTTGTC AGTTTGTTTG

1951 CATGAGGTGG CATACTGGTA CATTCTAAGC ATTGGAGCAC AGACTGACTT

2001 CCTTTCTGTC TTCTTCTCTG GATATACCTT CAAACACAAA ATGGTCTATG

2051 AAGACACACT CACCCTATTC CCATTCTCAG GAGAAACTGT CTTCATGTCG

2101 ATGGAAAACC CAGGTCTATG GATTCTGGGG TGCCACAACT CAGACTTTCG

2151 GAACAGAGGC ATGACCGCCT TACTGAAGGT TTCTAGTTGT GACAAGAACA

2201 CTGGTGATTA TTACGAGGAC AGTTATGAAG ATATTTCAGC ATACTTGCTG

2251 AGTAAAAACA ATGCCATTGA ACCAAGAAGC TTCTCTCAAA ACCCACCAGT

2301 CTTGAAACGC CATCAACGGG AAATAACTCG TACTACTCTT CAGTCAGATC

2351 AAGAGGAAAT TGACTATGAT GATACCATAT CAGTTGAAAT GAAGAAGGAA 2401 GATTTTGACA TTTATGATGA GGATGAAAAT CAGAGCCCCC

GCAGCTTTCA

2451 AAAGAAAACA CGACACTATT TTATTGCTGC AGTGGAGAGG

CTCTGGGATT

2501 ATGGGATGAG TAGCTCCCCA CATGTTCTAA GAAACAGGGC

TCAGAGTGGC

2551 AGTGTCCCTC AGTTCAAGAA AGTTGTTTTC CAGGAATTTA

CTGATGGCTC

2601 CTTTACTCAG CCCTTATACC GTGGAGAACT AAATGAACAT

TTGGGACTCC

2651 TGGGGCCATA TATAAGAGCA GAAGTTGAAG ATAATATCAT

GGTAACTTTC

2701 AGAAATCAGG CCTCTCGTCC CTATTCCTTC TATTCTAGCC

TTATTTCTTA

2751 TGAGGAAGAT CAGAGGCAAG GAGCAGAACC TAGAAAAAAC

TTTGTCAAGC

2801 CTAATGAAAC CAAAACTTAC TTTTGGAAAG TGCAACATCA

TATGGCACCC

2851 AC AAAGATG AGTTTGACTG CAAAGCCTGG GCTTATTTCT

CTGATGTTGA

2901 CCTGGAAAAA GATGTGCACT CAGGCCTGAT TGGACCCCTT

CTGGTCTGCC

2951 ACAC AACAC ACTGAACCCT GCTCATGGGA GACAAGTGAC

AGTACAGGAA

3001 TTTGCTCTGT TTTTCACCAT CTTTGATGAG ACCAAAAGCT

GGTACTTCAC

3051 TGAAAA A G GAAAGAAACT GCAGGGCTCC CTGCAATATC

CAGATGGAAG

3101 ATCCCACTTT TAAAGAGAAT TATCGCTTCC ATGCAATCAA

TGGCTACATA

3151 ATGGATACAC TACCTGGCTT AGTAATGGCT CAGGATCAAA

GGATTCGATG

3201 GTATCTGCTC AGCATGGGCA GCAATGAAAA CATCCATTCT

ATTCATTTCA

3251 GTGGACATGT GTTCACTGTA CGAAAAAAAG AGGAGTATAA

AATGGCACTG

3301 TACAATCTCT ATCCAGGTGT TTTTGAGACA GTGGAAATGT

TACCATCCAA

3351 AGCTGGAATT TGGCGGGTGG AATGCCTTAT TGGCGAGCAT CTACATGCTG

3401 GGATGAGCAC ACTTTTTCTG GTGTACAGCA ATAAGTGTCA

GACTCCCCTG

3451 GGAATGGCTT CTGGACACAT TAGAGATTTT CAGATTACAG

CTTCAGGACA

3501 ATATGGACAG TGGGCCCCAA AGCTGGCCAG ACTTCATTAT

TCCGGATCAA

3551 TCAATGCCTG GAGCACCAAG GAGCCCTTTT CTTGGATCAA GGTGGATCTG

3601 TTGGCACCAA TGATTATTCA CGGCATCAAG ACCCAGGGTG

CCCGTCAGAA 3651 GTTCTCCAGC CTCTACATCT CTCAGTTTAT CATCATGTAT AGTCTTGATG

3701 GGAAGAAGTG GCAGACTTAT CGAGGAAATT CCACTGGAAC

CTTAATGGTC

3751 TTCTTTGGCA ATGTGGATTC ATCTGGGATA AAACACAATA TTTTTAACCC

3801 TCCAATTATT GCTCGATACA TCCGTTTGCA CCCAACTCAT

TATAGCATTC

3851 GCAGCACTCT TCGCATGGAG TTGATGGGCT GTGATTTAAA TAGTTGCAGC

3901 ATGCCATTGG GAATGGAGAG TAAAGCAATA TCAGATGCAC

AGATTACTGC

3951 TTCATCCTAC TTTACCAATA TGTTTGCCAC CTGGTCTCCT TCAAAAGCTC

4001 GACTTCACCT CCAAGGGAGG AGTAATGCCT GGAGACCTCA GGTGAATAAT

4051 CCAAAAGAGT GGCTGCAAGT GGACTTCCAG AAGACAATGA

AAGTCACAGG

4101 AGTAACTACT CAGGGAGTAA AATCTCTGCT TACCAGCATG TATGTGAAGG

4151 AGTTCCTCAT CTCCAGCAGT CAAGATGGCC ATCAGTGGAC TCTCTTTTTT

4201 CAGAATGGCA AAGTAAAGGT TTTTCAGGGA AATCAAGACT CCTTCACACC

4251 TGTGGTGAAC TCTCTAGACC CACCGTTACT GACTCGCTAC CTTCGAATTC

4301 ACCCCCAGAG TTGGGTGCAC CAGATTGCCC TGAGGATGGA GGTTCTGGGC

4351 TGCGAGGCAC AGGACCTCTA CGACAAAACT CACACATGCC CACCGTGCCC

4401 AGCTCCAGAA CTCCTGGGCG GACCGTCAGT CTTCCTCTTC CCCCCAAAAC

4451 CCAAGGACAC CCTCATGATC TCCCGGACCC CTGAGGTCAC ATGCGTGGTG

4501 GTGGACGTGA GCCACGAAGA CCCTGAGGTC AAGTTCAACT GGTACGTGGA

4551 CGGCGTGGAG GTGCATAATG CCAAGACAAA GCCGCGGGAG GAGCAGTACA

4601 ACAGCACGTA CCGTGTGGTC AGCGTCCTCA CCGTCCTGCA CCAGGACTGG

4651 CTGAATGGCA AGGAGTACAA GTGCAAGGTC TCCAACAAAG CCCTCCCAGC

4701 CCCCATCGAG AAAACCATCT CCAAAGCCAA AGGGCAGCCC CGAGAACCAC

4751 AGGTGTACAC CCTGCCCCCA TCCCGGGATG AGCTGACCAA GAACCAGGTC

4801 AGCCTGACCT GCCTGGTCAA AGGCTTCTAT CCCAGCGACA TCGCCGTGGA

4851 GTGGGAGAGC AATGGGCAGC CGGAGAACAA CTACAAGACC ACGCCTCCCG 4901 TGTTGGACTC CGACGGCTCC TTCTTCCTCT ACAGCAAGCT

CACCGTGGAC

4951 AAGAGCAGGT GGCAGCAGGG GAACGTCTTC TCATGCTCCG

TGATGCATGA

5001 GGCTCTGCAC AACCACTACA CGCAGAAGAG CCTCTCCCTG

TCTCCGGGTA

5051 AATGA pSYNFVIII 010 protein sequence-(Dual chain FVIIIFc) (SEP ID NO: 126)

1 MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL

GELPVDARFP

51 PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL

LGPTIQAEVY

101 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR

EKEDDKVFPG

151 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG

LIGALLVCRE

201 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD

AASARAWPKM

251 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL

EGHTFLVRNH

301 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME

AYVKVDSCPE

351 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI

RSVAKKHPKT

401 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR

KYKKVRFMAY

451 TDETFKTREA IQHESGILGP LLYGEVGDTL LI IFKNQASR

PYNIYPHGIT

501 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP

TKSDPRCLTR

551 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR

NVILFSVFDE

601 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHS INGYV

FDSLQLSVCL

651 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF

PFSGETVFMS

701 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED

SYEDI SAYLL

751 SKNNAIEPRS FSQNPPVLKR HQREITRTTL QSDQEEIDYD

DTISVEMKKE

801 DFDIYDEDEN QSPRSFQKKT RHYFIAAVER LWDYGMSSSP

HVLRNRAQSG

851 SVPQFKKVVF QEFTDGSFTQ PLYRGELNEH LGLLGPYIRA

EVED IMVTF

901 RNQASRPYSF YSSLISYEED QRQGAEPRKN FVKPNETKTY

FWKVQHHMAP

951 TKDEFDCKAW AYFSDVDLEK DVHSGLIGPL LVCHTNTLNP

AHGRQVTVQE

1001 FALFFTIFDE TKSWYFTENM ERNCRAPCNI QMEDPTFKEN

YRFHAINGYI 1051 MDTLPGLVMA QDQRIRWYLL SMGSNENIHS IHFSGHVFTV

RKKEEYKMAL

1101 YNLYPGVFET VEMLPSKAGI WRVECLIGEH LHAGMSTLFL

VYSNKCQTPL

1151 GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK

EPFSWIKVDL

1201 LAPMI IHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY

RGNSTGTLMV

1251 FFGNVDSSGI KHNIFNPPII ARYIRLHPTH YSIRSTLRME

LMGCDLNSCS

1301 MPLGMESKAI SDAQITASSY FTNMFATWSP SKARLHLQGR

SNAWRPQVNN

1351 PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM YVKEFLISSS

QDGHQWTLFF

1401 QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH

QIALRMEVLG

1451 CEAQDLYDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI

SRTPEVTCVV

1501 VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV

SVLTVLHQDW

1551 LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP

SRDELTKNQV

1601 SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS

FFLYSKLTVD

1651 KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK*

Example 14: A new class of coagulation factor VIII molecules with greater than threefold half-life extension in hemophilia A mice

[0380] The new class of FVIII molecules was designed to contain two

polypeptides; one that consists of a single chain B-domain deleted (BDD) FVIII with XTEN inserted at one or more locations within the FVIII sequence, and one that is composed of the D'D3 region of VWF. Each polypeptide was also recombinantly fused to the Fc region of IgGl to enable the D'D3 region to be correctly aligned to bind the FVIII moiety. The resulting FVIII variants were expressed in HEK 293 cells by transient transfection, and purified from the conditioned media. FVIII activity was evaluated by FVIII chromogenic assay and the pharmacokinetic properties were assessed in both FVIII knockout (HemA) and FVIII/VWF double knock-out (DKO) mice.

[0381] Incorporating XTEN and D'D3 region of VWF into rFVIII led to the

uncoupling of the clearance of the fusion proteins from endogenous VWF while extending their circulating half-life. FVIII in this fusion configuration is completely shielded from interacting with VWF, as measured by biolayer interferometry (Octet) analysis. Consistent with this, their pharmacokinetic profiles in HemA and DKO mice were found to be identical, indicating that their clearance rate in mice was effectively disconnected from VWF. Optimization of

XTEN length and the locations for inserting XTEN identified a subset of the proteins that have exceeded the VWF limitation (16 hours), reaching a circulating half-life of up to 30 hours in HemA mice representing a 4-fold improvement over

BDD-FVIII. Importantly, these proteins maintained their functionality, as judged by FVIII chromogenic assay.

The VWF dependency has set a fundamental limitation for half-life of therapeutic FVIII. Uncoupling FVIII from VWF clearance pathways while extending half-life by the fusion of D'D3 region of VWF and XTEN has generated a novel FVIII molecule with a 4-fold half-life extension. This is the first report of an engineered FVIII that has exceeded the half-life limitation observed through industry-wide efforts in development of long-lasting FVIII, representing a potentially significant advancement in prophylactic treatment of hemophilia A.

Table 25: Protein sequences of FVIII-XTEN-Fc and VWF-Fc constructs FVIII 195 protein sequence (dual chain FVIIIFc with two 144 AE XTENs at amino acid 1656 and 1900) (SEP ID NO: 105)

1 MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL

GELPVDARFP

51 PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL

LGPTIQAEVY

101 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR

EKEDDKVFPG

151 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG

LIGALLVCRE

201 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD

AASARAWPKM

251 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL

EGHTFLVRNH

301 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME

AYVKVDSCPE

351 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI

RSVAKKHPKT

401 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR

KYKKVRFMAY

451 TDETFKTREA IQHESGILGP LLYGEVGDTL LI IFKNQASR

PYNIYPHGIT

501 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP

TKSDPRCLTR

551 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR

NVILFSVFDE 601 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHS INGYV

FDSLQLSVCL

651 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF

PFSGETVFMS

701 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED

SYEDI SAYLL

751 SKNNAIEPRS FSQNPPVLKR HQREITRTTL QGAPGTPGSG

TASSSPGASP

801 GTSSTGSPGA SPGTSSTGSP GASPGTSSTG SPGSSPSAST

GTGPGTPGSG

851 TASSSPGASP GTSSTGSPGA SPGTSSTGSP GASPGTSSTG

SPGSSTPSGA

901 TGSPGSSTPS GATGSPGASP GTSSTGSPAS SSDQEEIDYD

D ISVEMKKE

951 DFDIYDEDEN QSPRSFQKKT RHYFIAAVER LWDYGMSSSP

HVLRNRAQSG

1001 SVPQFKKVVF QEFTDGSFTQ PLYRGELNEH LGLLGPYIRA

EVED IMVTF

1051 RNQASRPYSF YSSLISYEED QRQGAEPRKN FVKPNETKTY

FWKVQHHMAP

1101 TKDEFDCKAW AYFSDVDLEK DVHSGLIGPL LVCHTNTLNP

AHGRQVTVQE

1151 FALFF IFDE TKSWYFTENM ERNCRGAPTS ESATPESGPG

SEPATSGSET

1201 PGTSESATPE SGPGSEPATS GSETPGTSES ATPESGPGTS

TEPSEGSAPG

1251 TSESATPESG PGSPAGSPTS TEEGSPAGSP TSTEEGSPAG

SPTSTEEGTS

1301 ESATPESGPG TSTEPSEGSA PGASSAPCNI QMEDPTFKEN

YRFHAINGYI

1351 MDTLPGLVMA QDQRIRWYLL SMGSNENIHS IHFSGHVFTV

RKKEEYKMAL

1401 YNLYPGVFET VEMLPSKAGI WRVECLIGEH LHAGMSTLFL

VYSNKCQTPL

1451 GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK

EPFSWIKVDL

1501 LAPMI IHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY

RGNSTGTLMV

1551 FFGNVDSSGI KHNIFNPPII ARYIRLHPTH YSIRSTLRME

LMGCDLNSCS

1601 MPLGMESKAI SDAQITASSY FTNMFATWSP SKARLHLQGR

SNAWRPQVNN

1651 PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM YVKEFLISSS

QDGHQWTLFF

1701 QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH

QIALRMEVLG

1751 CEAQDLYDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI

SRTPEVTCVV

1801 VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV

SVLTVLHQDW 1851 LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP

SRDELTKNQV

1901 SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS

FFLYSKLTVD

1951 KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK*

FVIII 196 protein sequence (dual chain FVIIIFc with three 144 AE XTENs at amino acid 26, 1656 and 1900) (SEP ID NO: 106)

1 MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL

GELPVGAPGS

51 SPSASTGTGP GSSPSASTGT GPGASPGTSS TGSPGASPGT

SSTGSPGSST

101 PSGATGSPGS SPSASTGTGP GASPGTSSTG SPGSSPSAST

GTGPGTPGSG

151 TASSSPGSST PSGATGSPGS STPSGATGSP GASPGTSSTG

SPASSDARFP

201 PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL

LGPTIQAEVY

251 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR

EKEDDKVFPG

301 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG

LIGALLVCRE

351 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD

AASARAWPKM

401 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL

EGHTFLVRNH

451 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME

AYVKVDSCPE

501 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI

RSVAKKHPKT

551 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR

KYKKVRFMAY

601 TDETFKTREA IQHESGILGP LLYGEVGDTL LI IFKNQASR

PYNIYPHGIT

651 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP

TKSDPRCLTR

701 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR

NVILFSVFDE

751 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHS INGYV

FDSLQLSVCL

801 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF

PFSGETVFMS

851 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED

SYEDI SAYLL

901 SKNNAIEPRS FSQNPPVLKR HQREITRTTL QGAPGTPGSG

TASSSPGASP

951 GTSSTGSPGA SPGTSSTGSP GASPGTSSTG SPGSSPSAST

GTGPGTPGSG

1001 TASSSPGASP GTSSTGSPGA SPGTSSTGSP GASPGTSSTG

SPGSSTPSGA 1051 TGSPGSSTPS GATGSPGASP GTSSTGSPAS SSDQEEIDYD

D ISVEMKKE

1101 DFDIYDEDEN QSPRSFQKKT RHYFIAAVER LWDYGMSSSP

HVLRNRAQSG

1151 SVPQFKKVVF QEFTDGSFTQ PLYRGELNEH LGLLGPYIRA

EVED IMVTF

1201 RNQASRPYSF YSSLISYEED QRQGAEPRKN FVKPNETKTY

FWKVQHHMAP

1251 TKDEFDCKAW AYFSDVDLEK DVHSGLIGPL LVCHTNTLNP

AHGRQVTVQE

1301 FALFFTIFDE TKSWYFTENM ERNCRGAPTS ESATPESGPG

SEPATSGSET

1351 PGTSESATPE SGPGSEPATS GSETPGTSES ATPESGPGTS

TEPSEGSAPG

1401 TSESATPESG PGSPAGSPTS TEEGSPAGSP TSTEEGSPAG

SPTSTEEGTS

1451 ESATPESGPG TSTEPSEGSA PGASSAPCNI QMEDPTFKEN

YRFHAINGYI

1501 MDTLPGLVMA QDQRIRWYLL SMGSNENIHS IHFSGHVFTV

RKKEEYKMAL

1551 YNLYPGVFET VEMLPSKAGI WRVECLIGEH LHAGMSTLFL

VYSNKCQTPL

1601 GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK

EPFSWIKVDL

1651 LAPMI IHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY

RGNSTGTLMV

1701 FFGNVDSSGI KHNIFNPPII ARYIRLHPTH YSIRSTLRME

LMGCDLNSCS

1751 MPLGMESKAI SDAQI ASSY FTNMFATWSP SKARLHLQGR

SNAWRPQVNN

1801 PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM YVKEFLISSS

QDGHQWTLFF

1851 QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH

QIALRMEVLG

1901 CEAQDLYDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI

SRTPEVTCVV

1951 VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV

SVLTVLHQDW

2001 LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP

SRDELTKNQV

2051 SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS

FFLYSKLTVD

2101 KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK*

FVIII 199 protein sequence (single chain FVIIIFc with three 144 AE XTENs at amino acid 1656 and 1900) (SEP ID NO: 107)

1 MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL

GELPVDARFP

51 PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL

LGPTIQAEVY 101 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR

EKEDDKVFPG

151 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG

LIGALLVCRE

201 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD

AASARAWPKM

251 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL

EGHTFLVRNH

301 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME

AYVKVDSCPE

351 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI

RSVAKKHPKT

401 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR

KYKKVRFMAY

451 TDETFKTREA IQHESGILGP LLYGEVGDTL LI IFKNQASR

PYNIYPHGIT

501 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP

TKSDPRCLTR

551 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR

NVILFSVFDE

601 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHS INGYV

FDSLQLSVCL

651 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF

PFSGETVFMS

701 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED

SYEDI SAYLL

751 SKNNAIEPRS FSQNPPVLKR HQAEITRTTL QGAPGTPGSG

TASSSPGASP

801 GTSSTGSPGA SPGTSSTGSP GASPGTSSTG SPGSSPSAST

GTGPGTPGSG

851 TASSSPGASP GTSSTGSPGA SPGTSSTGSP GASPGTSSTG

SPGSSTPSGA

901 TGSPGSSTPS GATGSPGASP GTSSTGSPAS SSDQEEIDYD

DTISVEMKKE

951 DFDIYDEDEN QSPRSFQKKT RHYFIAAVER LWDYGMSSSP

HVLRNRAQSG

1001 SVPQFKKVVF QEFTDGSFTQ PLYRGELNEH LGLLGPYIRA

EVED IMVTF

1051 RNQASRPYSF YSSLISYEED QRQGAEPRKN FVKPNETKTY

FWKVQHHMAP

1101 TKDEFDCKAW AYFSDVDLEK DVHSGLIGPL LVCHTNTLNP

AHGRQVTVQE

1151 FALFFTIFDE TKSWYFTENM ERNCRGAPTS ESATPESGPG

SEPATSGSET

1201 PGTSESATPE SGPGSEPATS GSETPGTSES ATPESGPGTS

TEPSEGSAPG

1251 TSESATPESG PGSPAGSPTS TEEGSPAGSP TSTEEGSPAG

SPTSTEEGTS

1301 ESATPESGPG TSTEPSEGSA PGASSAPCNI QMEDPTFKEN

YRFHAINGYI 1351 MDTLPGLVMA QDQRIRWYLL SMGSNENIHS IHFSGHVFTV

RKKEEYKMAL

1401 YNLYPGVFET VEMLPSKAGI WRVECLIGEH LHAGMSTLFL

VYSNKCQTPL

1451 GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK

EPFSWIKVDL

1501 LAPMI IHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY

RGNSTGTLMV

1551 FFGNVDSSGI KHNIFNPPII ARYIRLHPTH YSIRSTLRME

LMGCDLNSCS

1601 MPLGMESKAI SDAQITASSY FTNMFATWSP SKARLHLQGR

SNAWRPQVNN

1651 PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM YVKEFLISSS

QDGHQWTLFF

1701 QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH

QIALRMEVLG

1751 CEAQDLYDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI

SRTPEVTCVV

1801 VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV

SVLTVLHQDW

1851 LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP

SRDELTKNQV

1901 SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS

FFLYSKLTVD

1951 KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK*

FVIII 201 protein sequence (single chain FVIIIFc with three 144 AE XTENs at amino acid 26, 1656 &1900) (SEP ID NO: 108)

1 MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL

GELPVGAPGS

51 SPSASTGTGP GSSPSASTGT GPGASPGTSS TGSPGASPGT

SSTGSPGSST

101 PSGATGSPGS SPSASTGTGP GASPGTSSTG SPGSSPSAST

GTGPGTPGSG

151 TASSSPGSST PSGATGSPGS STPSGATGSP GASPGTSSTG

SPASSDARFP

201 PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL

LGPTIQAEVY

251 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR

EKEDDKVFPG

301 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG

LIGALLVCRE

351 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD

AASARAWPKM

401 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL

EGHTFLVRNH

451 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME

AYVKVDSCPE

501 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI

RSVAKKHPKT 551 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR

KYKKVRFMAY

601 TDETFKTREA IQHESGILGP LLYGEVGDTL LI IFKNQASR

PYNIYPHGIT

651 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP

TKSDPRCLTR

701 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR

NVILFSVFDE

751 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHS INGYV

FDSLQLSVCL

801 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF

PFSGETVFMS

851 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED

SYEDI SAYLL

901 SKNNAIEPRS FSQNPPVLKR HQAEITRTTL QGAPGTPGSG

TASSSPGASP

951 GTSSTGSPGA SPGTSSTGSP GASPGTSSTG SPGSSPSAST

GTGPGTPGSG

1001 TASSSPGASP GTSSTGSPGA SPGTSSTGSP GASPGTSSTG

SPGSSTPSGA

1051 TGSPGSSTPS GATGSPGASP GTSSTGSPAS SSDQEEIDYD

D ISVEMKKE

1101 DFDIYDEDEN QSPRSFQKKT RHYFIAAVER LWDYGMSSSP

HVLRNRAQSG

1151 SVPQFKKVVF QEFTDGSFTQ PLYRGELNEH LGLLGPYIRA

EVED IMVTF

1201 RNQASRPYSF YSSLISYEED QRQGAEPRKN FVKPNETKTY

FWKVQHHMAP

1251 TKDEFDCKAW AYFSDVDLEK DVHSGLIGPL LVCHTNTLNP

AHGRQVTVQE

1301 FALFF IFDE TKSWYFTENM ERNCRGAPTS ESATPESGPG

SEPATSGSET

1351 PGTSESATPE SGPGSEPATS GSETPGTSES ATPESGPGTS

TEPSEGSAPG

1401 TSESATPESG PGSPAGSPTS TEEGSPAGSP TSTEEGSPAG

SPTSTEEGTS

1451 ESATPESGPG TSTEPSEGSA PGASSAPCNI QMEDPTFKEN

YRFHAINGYI

1501 MDTLPGLVMA QDQRIRWYLL SMGSNENIHS IHFSGHVFTV

RKKEEYKMAL

1551 YNLYPGVFET VEMLPSKAGI WRVECLIGEH LHAGMSTLFL

VYSNKCQTPL

1601 GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK

EPFSWIKVDL

1651 LAPMI IHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY

RGNSTGTLMV

1701 FFGNVDSSGI KHNIFNPPII ARYIRLHPTH YSIRSTLRME

LMGCDLNSCS

1751 MPLGMESKAI SDAQITASSY FTNMFATWSP SKARLHLQGR

SNAWRPQVNN 1801 PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM YVKEFLISSS QDGHQWTLFF

1851 QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH

QIALRMEVLG

1901 CEAQDLYDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI

SRTPEVTCVV

1951 VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV

SVLTVLHQDW

2001 LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP

SRDELTKNQV

2051 SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS

FFLYSKLTVD

2101 KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK*

FVIII 203 protein sequence (single chain FVIIIFc with two AE XTENs; one 288AE XTEN

in B-domain and one 144 AE XTEN at amino acid 1900) (SEP ID NO: 109)

1 MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL GELPVDARFP

51 PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY

101 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR

EKEDDKVFPG

151 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG

LIGALLVCRE

201 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD

AASARAWPKM

251 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL

EGHTFLVRNH

301 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME

AYVKVDSCPE

351 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI

RSVAKKHPKT

401 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR

KYKKVRFMAY

451 TDETFKTREA IQHESGILGP LLYGEVGDTL LI IFKNQASR

PYNIYPHGIT

501 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP

TKSDPRCLTR

551 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR

NVILFSVFDE

601 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHS INGYV

FDSLQLSVCL

651 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF

PFSGETVFMS

701 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED

SYEDI SAYLL

751 SKNNAIEPRS FSQNGAPGTS ESATPESGPG SEPATSGSET

PGTSESATPE

801 SGPGSEPATS GSETPGTSES ATPESGPGTS TEPSEGSAPG

SPAGSPTSTE 851 EGTSESATPE SGPGSEPATS GSETPGTSES ATPESGPGSP

AGSPTSTEEG

901 SPAGSPTSTE EGTSTEPSEG SAPGTSESAT PESGPGTSES

ATPESGPGTS

951 ESATPESGPG SEPATSGSET PGSEPATSGS ETPGSPAGSP

TSTEEGTSTE

1001 PSEGSAPGTS TEPSEGSAPG SEPATSGSET PGTSESATPE

SGPGTSTEPS

1051 EGSAPASSPP VLKRHQAEIT RTTLQSDQEE IDYDDTISVE

MKKEDFDIYD

1101 EDENQSPRSF QKKTRHYFIA AVERLWDYGM SSSPHVLRNR

AQSGSVPQFK

1151 KVVFQEFTDG SFTQPLYRGE LNEHLGLLGP YIRAEVEDNI

MVTFRNQASR

1201 PYSFYSSLIS YEEDQRQGAE PRKNFVKPNE TKTYFWKVQH

HMAPTKDEFD

1251 CKAWAYFSDV DLEKDVHSGL IGPLLVCHTN TLNPAHGRQV

TVQEFALFFT

1301 IFDETKSWYF TENMERNCRG APTSESATPE SGPGSEPATS

GSETPGTSES

1351 ATPESGPGSE PATSGSETPG TSESATPESG PGTSTEPSEG

SAPGTSESAT

1401 PESGPGSPAG SPTSTEEGSP AGSPTSTEEG SPAGSPTSTE

EGTSESATPE

1451 SGPGTSTEPS EGSAPGASSA PCNIQMEDPT FKENYRFHAI

NGYIMDTLPG

1501 LVMAQDQRIR WYLLSMGSNE NIHSIHFSGH VFTVRKKEEY

KMALYNLYPG

1551 VFETVEMLPS KAGIWRVECL IGEHLHAGMS TLFLVYSNKC

QTPLGMASGH

1601 IRDFQITASG QYGQWAPKLA RLHYSGSINA WSTKEPFSWI

KVDLLAPMI I

1651 HGIKTQGARQ KFSSLYISQF I IMYSLDGKK WQTYRGNSTG

TLMVFFGNVD

1701 SSGIKHNIFN PPIIARYIRL HPTHYS IRST LRMELMGCDL

NSCSMPLGME

1751 SKAISDAQIT ASSYFTNMFA TWSPSKARLH LQGRSNAWRP

QVNNPKEWLQ

1801 VDFQKTMKVT GVTTQGVKSL LTSMYVKEFL ISSSQDGHQW

TLFFQNGKVK

1851 VFQGNQDSFT PVVNSLDPPL LTRYLRIHPQ SWVHQIALRM

EVLGCEAQDL

1901 YDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMI SRTPEV

TCVVVDVSHE

1951 DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL

HQDWLNGKEY

2001 KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT

KNQVSLTCLV

2051 KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK

LTVDKSRWQQ

2101 GNVFSCSVMH EALHNHYTQK SLSLSPGK* FVIII 204 protein sequence (single chain FVIIIFc with two AE XTENs; one 288AE XTEN

in B-domain and one 144 AE XTEN at amino acid 403) (SEP ID NO: 110)

1 MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL GELPVDARFP

51 PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY

101 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR

EKEDDKVFPG

151 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG

LIGALLVCRE

201 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD

AASARAWPKM

251 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL

EGHTFLVRNH

301 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME

AYVKVDSCPE

351 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI

RSVAKKHPKT

401 WVHYIAAEEE DWDYAPLVLA PDGAPTSTEP SEGSAPGSPA

GSPTSTEEGT

451 STEPSEGSAP GTSTEPSEGS APGTSESATP ESGPGTSTEP

SEGSAPGTSE

501 SATPESGPGS EPATSGSETP GTSTEPSEGS APGTSTEPSE

GSAPGTSESA

551 TPESGPGTSE SATPESGPGA SSDRSYKSQY LNNGPQRIGR

KYKKVRFMAY

601 TDETFKTREA IQHESGILGP LLYGEVGDTL LI IFKNQASR

PYNIYPHGIT

651 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP

TKSDPRCLTR

701 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR

NVILFSVFDE

751 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHS INGYV

FDSLQLSVCL

801 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF

PFSGETVFMS

851 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED

SYEDI SAYLL

901 SKNNAIEPRS FSQNGAPGTS ESATPESGPG SEPATSGSET

PGTSESATPE

951 SGPGSEPATS GSETPGTSES ATPESGPGTS TEPSEGSAPG

SPAGSPTSTE

1001 EGTSESATPE SGPGSEPATS GSETPGTSES ATPESGPGSP

AGSPTSTEEG

1051 SPAGSPTSTE EGTSTEPSEG SAPGTSESAT PESGPGTSES

ATPESGPGTS

1101 ESATPESGPG SEPATSGSET PGSEPATSGS ETPGSPAGSP

TSTEEGTSTE 1151 PSEGSAPGTS TEPSEGSAPG SEPATSGSET PGTSESATPE

SGPGTSTEPS

1201 EGSAPASSPP VLKRHQAEIT RTTLQSDQEE IDYDDTISVE

MKKEDFDIYD

1251 EDENQSPRSF QKKTRHYFIA AVERLWDYGM SSSPHVLRNR

AQSGSVPQFK

1301 KVVFQEFTDG SFTQPLYRGE LNEHLGLLGP YIRAEVEDNI

MVTFRNQASR

1351 PYSFYSSLIS YEEDQRQGAE PRKNFVKPNE TKTYFWKVQH

HMAPTKDEFD

1401 CKAWAYFSDV DLEKDVHSGL IGPLLVCHTN TLNPAHGRQV

TVQEFALFFT

1451 IFDETKSWYF TENMERNCRA PCNIQMEDPT FKENYRFHAI

NGYIMDTLPG

1501 LVMAQDQRIR WYLLSMGSNE NIHS IHFSGH VFTVRKKEEY

KMALYNLYPG

1551 VFETVEMLPS KAGIWRVECL IGEHLHAGMS TLFLVYSNKC

QTPLGMASGH

1601 IRDFQITASG QYGQWAPKLA RLHYSGS INA WSTKEPFSWI

KVDLLAPMI I

1651 HGIKTQGARQ KFSSLYISQF I IMYSLDGKK WQTYRGNSTG

TLMVFFGNVD

1701 SSGIKHNIFN PPIIARYIRL HPTHYS IRST LRMELMGCDL

NSCSMPLGME

1751 SKAISDAQIT ASSYFTNMFA TWSPSKARLH LQGRSNAWRP

QVNNPKEWLQ

1801 VDFQKTMKVT GVTTQGVKSL LTSMYVKEFL ISSSQDGHQW

TLFFQNGKVK

1851 VFQGNQDSFT PVVNSLDPPL LTRYLRIHPQ SWVHQIALRM

EVLGCEAQDL

1901 YDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMI SRTPEV

TCVVVDVSHE

1951 DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL

HQDWLNGKEY

2001 KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT

KNQVSLTCLV

2051 KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK

LTVDKSRWQQ

2101 GNVFSCSVMH EALHNHYTQK SLSLSPGK*

FVIII 205 protein sequence (single chain FVIIIFc with two AE XTENs; one 288AE XTEN in B-domain and one 144 AE XTEN at amino acid 18) (SEP ID NP: 111)

1 MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQGAP TSESATPESG

51 PGSEPATSGS ETPGTSESAT PESGPGSEPA TSGSETPGTS

ESATPESGPG

101 TSTEPSEGSA PGSPAGSPTS TEEGTSESAT PESGPGSEPA

TSGSETPGTS 151 ESATPESGPG SPAGSPTSTE EGSPAGSPTS TEEGASSSDL

GELPVDARFP

201 PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL

LGPTIQAEVY

251 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR

EKEDDKVFPG

301 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG

LIGALLVCRE

351 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD

AASARAWPKM

401 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL

EGHTFLVRNH

451 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME

AYVKVDSCPE

501 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI

RSVAKKHPKT

551 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR

KYKKVRFMAY

601 TDETFKTREA IQHESGILGP LLYGEVGDTL LI IFKNQASR

PYNIYPHGIT

651 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP

TKSDPRCLTR

701 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR

NVILFSVFDE

751 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHS INGYV

FDSLQLSVCL

801 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF

PFSGETVFMS

851 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED

SYEDI SAYLL

901 SKNNAIEPRS FSQNGAPGTS ESATPESGPG SEPATSGSET

PGTSESATPE

951 SGPGSEPATS GSETPGTSES ATPESGPGTS TEPSEGSAPG

SPAGSPTSTE

1001 EGTSESATPE SGPGSEPATS GSETPGTSES ATPESGPGSP

AGSPTSTEEG

1051 SPAGSPTSTE EGTSTEPSEG SAPGTSESAT PESGPGTSES

ATPESGPGTS

1101 ESATPESGPG SEPATSGSET PGSEPATSGS ETPGSPAGSP

TSTEEGTSTE

1151 PSEGSAPGTS TEPSEGSAPG SEPATSGSET PGTSESATPE

SGPGTSTEPS

1201 EGSAPASSPP VLKRHQAEIT RTTLQSDQEE IDYDDTISVE

MKKEDFDIYD

1251 EDENQSPRSF QKKTRHYFIA AVERLWDYGM SSSPHVLRNR

AQSGSVPQFK

1301 KVVFQEFTDG SFTQPLYRGE LNEHLGLLGP YIRAEVEDNI

MVTFRNQASR

1351 PYSFYSSLIS YEEDQRQGAE PRKNFVKPNE TKTYFWKVQH

HMAPTKDEFD 1401 CKAWAYFSDV DLEKDVHSGL IGPLLVCHTN TLNPAHGRQV TVQEFALFFT

1451 IFDETKSWYF TENMERNCRA PCNIQMEDPT FKENYRFHAI

NGYIMDTLPG

1501 LVMAQDQRIR WYLLSMGSNE NIHSIHFSGH VFTVRKKEEY

KMALYNLYPG

1551 VFETVEMLPS KAGIWRVECL IGEHLHAGMS TLFLVYSNKC

QTPLGMASGH

1601 IRDFQITASG QYGQWAPKLA RLHYSGS INA WSTKEPFSWI

KVDLLAPMI I

1651 HGIKTQGARQ KFSSLYISQF I IMYSLDGKK WQTYRGNSTG

TLMVFFGNVD

1701 SSGIKHNIFN PPIIARYIRL HPTHYS IRST LRMELMGCDL

NSCSMPLGME

1751 SKAISDAQIT ASSYFTNMFA TWSPSKARLH LQGRSNAWRP

QVNNPKEWLQ

1801 VDFQKTMKVT GVTTQGVKSL LTSMYVKEFL ISSSQDGHQW

TLFFQNGKVK

1851 VFQGNQDSFT PVVNSLDPPL LTRYLRIHPQ SWVHQIALRM

EVLGCEAQDL

1901 YDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMI SRTPEV

TCVVVDVSHE

1951 DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL

HQDWLNGKEY

2001 KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT

KNQVSLTCLV

2051 KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK

LTVDKSRWQQ

2101 GNVFSCSVMH EALHNHYTQK SLSLSPGK* pSYN FVIII 266 protein sequence (FVIII Fc with 42 AE-XTEN at amino acid 18 and 288 AE XTEN in B-domain) SEP ID NO: 112

1 MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQGAP GSPAGSPTST

51 EEGTSESATP ESGPGSEPAT SGSETPASSS DLGELPVDAR FPPRVPKSFP

101 FNTSVVYKKT LFVEFTDHLF NIAKPRPPWM GLLGPTIQAE

VYDTVVITLK

151 NMASHPVSLH AVGVSYWKAS EGAEYDDQTS QREKEDDKVF

PGGSHTYVWQ

201 VLKENGPMAS DPLCLTYSYL SHVDLVKDLN SGLIGALLVC

REGSLAKEKT

251 QTLHKFILLF AVFDEGKSWH SETKNSLMQD RDAASARAWP

KMHTVNGYVN

301 RSLPGLIGCH RKSVYWHVIG MGTTPEVHS I FLEGHTFLVR

NHRQASLEIS

351 PITFLTAQTL LMDLGQFLLF CHISSHQHDG MEAYVKVDSC

PEEPQLRMKN

401 NEEAEDYDDD LTDSEMDVVR FDDDNSPSFI QIRSVAKKHP

KTWVHYIAAE 451 EEDWDYAPLV LAPDDRSYKS QYLNNGPQRI GRKYKKVRFM

AYTDETFKTR

501 EAIQHESGIL GPLLYGEVGD TLLI IFKNQA SRPYNIYPHG

ITDVRPLYSR

551 RLPKGVKHLK DFPILPGEIF KYKWTVTVED GPTKSDPRCL

TRYYSSFVNM

601 ERDLASGLIG PLLICYKESV DQRGNQIMSD KRNVILFSVF

DENRSWYLTE

651 NIQRFLPNPA GVQLEDPEFQ ASNIMHSING YVFDSLQLSV

CLHEVAYWYI

701 LSIGAQTDFL SVFFSGYTFK HKMVYEDTLT LFPFSGETVF

MSMENPGLWI

751 LGCHNSDFRN RGMTALLKVS SCDKNTGDYY EDSYEDI SAY

LLSKNNAIEP

801 RSFSQNGAPG TSESATPESG PGSEPATSGS ETPGTSESAT

PESGPGSEPA

851 TSGSETPGTS ESATPESGPG TSTEPSEGSA PGSPAGSPTS

TEEGTSESAT

901 PESGPGSEPA TSGSETPGTS ESATPESGPG SPAGSPTSTE

EGSPAGSPTS

951 TEEGTSTEPS EGSAPGTSES ATPESGPGTS ESATPESGPG TSESATPESG

1001 PGSEPATSGS ETPGSEPATS GSETPGSPAG SPTSTEEGTS

TEPSEGSAPG

1051 TSTEPSEGSA PGSEPATSGS ETPGTSESAT PESGPGTSTE

PSEGSAPASS

1101 PPVLKRHQAE ITRTTLQSDQ EEIDYDDTIS VEMKKEDFDI

YDEDENQSPR

1151 SFQKKTRHYF IAAVERLWDY GMSSSPHVLR NRAQSGSVPQ

FKKVVFQEFT

1201 DGSFTQPLYR GELNEHLGLL GPYIRAEVED NIMVTFRNQA

SRPYSFYSSL

1251 ISYEEDQRQG AEPRKNFVKP NETKTYFWKV QHHMAPTKDE

FDCKAWAYFS

1301 DVDLEKDVHS GLIGPLLVCH TNTLNPAHGR QVTVQEFALF

FTIFDETKSW

1351 YFTENMERNC RAPCNIQMED PTFKENYRFH AINGYIMDTL

PGLVMAQDQR

1401 IRWYLLSMGS NENIHS IHFS GHVFTVRKKE EYKMALYNLY

PGVFETVEML

1451 PSKAGIWRVE CLIGEHLHAG MSTLFLVYSN KCQTPLGMAS

GHIRDFQITA

1501 SGQYGQWAPK LARLHYSGS I NAWSTKEPFS WIKVDLLAPM

I IHGIKTQGA

1551 RQKFSSLYIS QFI IMYSLDG KKWQTYRGNS TGTLMVFFGN

VDSSGIKHNI

1601 FNPPIIARYI RLHPTHYSIR STLRMELMGC DLNSCSMPLG

MESKAI SDAQ

1651 ITASSYFTNM FATWSPSKAR LHLQGRSNAW RPQVNNPKEW LQVDFQKTMK 1701 VTGVTTQGVK SLLTSMYVKE FLISSSQDGH QWTLFFQNGK VKVFQGNQDS

1751 FTPVVNSLDP PLLTRYLRIH PQSWVHQIAL RMEVLGCEAQ

DLYDKTHTCP

1801 PCPAPELLGG PSVFLFPPKP KDTLMI SRTP EVTCVVVDVS

HEDPEVKFNW

1851 YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK

EYKCKVSNKA

1901 LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC

LVKGFYPSDI

1951 AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW

QQGNVFSCSV

2001 MHEALHNHYT QKSLSLSPGK * pSYN FVIII 267 protein sequence (FVIII Fc with 72 AE-XTEN at amino acid 18 and 288

AE XTEN in B-domain) SEP ID NO: 113

1 MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQGAP TSESATPESG

51 PGSEPATSGS ETPGTSESAT PESGPGSEPA TSGSETPGTS ESATPESGPG

101 TSTEPSEGSA PGASSSDLGE LPVDARFPPR VPKSFPFNTS

VVYKKTLFVE

151 FTDHLFNIAK PRPPWMGLLG PTIQAEVYDT VVITLKNMAS

HPVSLHAVGV

201 SYWKASEGAE YDDQTSQREK EDDKVFPGGS HTYVWQVLKE

NGPMASDPLC

251 LTYSYLSHVD LVKDLNSGLI GALLVCREGS LAKEKTQTLH

KFILLFAVFD

301 EGKSWHSETK NSLMQDRDAA SARAWPKMHT VNGYVNRSLP

GLIGCHRKSV

351 YWHVIGMGTT PEVHSIFLEG HTFLVRNHRQ ASLEISPITF

LTAQTLLMDL

401 GQFLLFCHIS SHQHDGMEAY VKVDSCPEEP QLRMKNNEEA

EDYDDDLTDS

451 EMDVVRFDDD NSPSFIQIRS VAKKHPKTWV HYIAAEEEDW

DYAPLVLAPD

501 DRSYKSQYLN NGPQRIGRKY KKVRFMAYTD ETFKTREAIQ

HESGILGPLL

551 YGEVGDTLLI IFKNQASRPY NIYPHGITDV RPLYSRRLPK

GVKHLKDFPI

601 LPGEIFKYKW TVTVEDGPTK SDPRCLTRYY SSFVNMERDL

ASGLIGPLLI

651 CYKESVDQRG NQIMSDKRNV ILFSVFDENR SWYLTENIQR

FLPNPAGVQL

701 EDPEFQASNI MHS INGYVFD SLQLSVCLHE VAYWYILSIG

AQTDFLSVFF

751 SGYTFKHKMV YEDTLTLFPF SGETVFMSME NPGLWILGCH

NSDFRNRGMT

801 ALLKVSSCDK NTGDYYEDSY EDISAYLLSK NNAIEPRSFS

QNGAPGTSES 851 ATPESGPGSE PATSGSETPG TSESATPESG PGSEPATSGS

ETPGTSESAT

901 PESGPGTSTE PSEGSAPGSP AGSPTSTEEG TSESATPESG

PGSEPATSGS

951 ETPGTSESAT PESGPGSPAG SPTSTEEGSP AGSPTSTEEG

TSTEPSEGSA

1001 PGTSESATPE SGPGTSESAT PESGPGTSES ATPESGPGSE

PATSGSETPG

1051 SEPATSGSET PGSPAGSPTS TEEGTSTEPS EGSAPGTSTE

PSEGSAPGSE

1101 PATSGSETPG TSESATPESG PGTSTEPSEG SAPASSPPVL

KRHQAEITRT

1151 TLQSDQEEID YDDTISVEMK KEDFDIYDED ENQSPRSFQK

KTRHYFIAAV

1201 ERLWDYGMSS SPHVLRNRAQ SGSVPQFKKV VFQEFTDGSF

TQPLYRGELN

1251 EHLGLLGPYI RAEVEDNIMV TFRNQASRPY SFYSSLISYE

EDQRQGAEPR

1301 KNFVKPNETK TYFWKVQHHM APTKDEFDCK AWAYFSDVDL

EKDVHSGLIG

1351 PLLVCHTNTL NPAHGRQVTV QEFALFFTIF DETKSWYFTE

NMERNCRAPC

1401 NIQMEDPTFK ENYRFHAING YIMDTLPGLV MAQDQRIRWY

LLSMGSNENI

1451 HSIHFSGHVF TVRKKEEYKM ALYNLYPGVF ETVEMLPSKA

GIWRVECLIG

1501 EHLHAGMSTL FLVYSNKCQT PLGMASGHIR DFQITASGQY

GQWAPKLARL

1551 HYSGSINAWS TKEPFSWIKV DLLAPMI IHG IKTQGARQKF

SSLYISQFII

1601 MYSLDGKKWQ TYRGNSTGTL MVFFGNVDSS GIKHNIFNPP

I IARYIRLHP

1651 THYS IRSTLR MELMGCDLNS CSMPLGMESK AISDAQITAS

SYFTNMFATW

1701 SPSKARLHLQ GRSNAWRPQV NNPKEWLQVD FQKTMKVTGV

TTQGVKSLLT

1751 SMYVKEFLIS SSQDGHQWTL FFQNGKVKVF QGNQDSFTPV

VNSLDPPLLT

1801 RYLRIHPQSW VHQIALRMEV LGCEAQDLYD KTHTCPPCPA

PELLGGPSVF

1851 LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG

VEVHNAKTKP

1901 REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP

IEKTISKAKG

1951 QPREPQVYTL PPSRDELTKN QVSLTCLVKG FYPSDIAVEW

ESNGQPENNY

2001 KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA

LHNHYTQKSL

2051 SLSPGK* pSYN FVIII 268 protein sequence (FVIII Fc with 144 AE-XTEN at amino acid 18) SEP ID NO: 114

1 MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQGAP TSESATPESG

51 PGSEPATSGS ETPGTSESAT PESGPGSEPA TSGSETPGTS ESATPESGPG

101 TSTEPSEGSA PGSPAGSPTS TEEGTSESAT PESGPGSEPA

TSGSETPGTS

151 ESATPESGPG SPAGSPTSTE EGSPAGSPTS TEEGASSSDL

GELPVDARFP

201 PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL

LGPTIQAEVY

251 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR

EKEDDKVFPG

301 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG

LIGALLVCRE

351 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD

AASARAWPKM

401 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL

EGHTFLVRNH

451 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME

AYVKVDSCPE

501 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI

RSVAKKHPKT

551 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR

KYKKVRFMAY

601 TDETFKTREA IQHESGILGP LLYGEVGDTL LI IFKNQASR

PYNIYPHGIT

651 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP

TKSDPRCLTR

701 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR

NVILFSVFDE

751 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHS INGYV

FDSLQLSVCL

801 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF

PFSGETVFMS

851 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED

SYEDI SAYLL

901 SKNNAIEPRS FSQNPPVLKR HQAEITRTTL QSDQEEIDYD

DTISVEMKKE

951 DFDIYDEDEN QSPRSFQKKT RHYFIAAVER LWDYGMSSSP

HVLRNRAQSG

1001 SVPQFKKVVF QEFTDGSFTQ PLYRGELNEH LGLLGPYIRA

EVED IMVTF

1051 RNQASRPYSF YSSLISYEED QRQGAEPRKN FVKPNETKTY

FWKVQHHMAP

1101 TKDEFDCKAW AYFSDVDLEK DVHSGLIGPL LVCHTNTLNP

AHGRQVTVQE

1151 FALFFTIFDE TKSWYFTENM ERNCRAPCNI QMEDPTFKEN

YRFHAINGYI 1201 MDTLPGLVMA QDQRIRWYLL SMGSNENIHS IHFSGHVFTV

RKKEEYKMAL

1251 YNLYPGVFET VEMLPSKAGI WRVECLIGEH LHAGMSTLFL

VYSNKCQTPL

1301 GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK

EPFSWIKVDL

1351 LAPMI IHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY

RGNSTGTLMV

1401 FFGNVDSSGI KHNIFNPPII ARYIRLHPTH YSIRSTLRME

LMGCDLNSCS

1451 MPLGMESKAI SDAQITASSY FTNMFATWSP SKARLHLQGR

SNAWRPQVNN

1501 PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM YVKEFLISSS

QDGHQWTLFF

1551 QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH

QIALRMEVLG

1601 CEAQDLYDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI

SRTPEVTCVV

1651 VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV

SVLTVLHQDW

1701 LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP

SRDELTKNQV

1751 SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS

FFLYSKLTVD

1801 KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK* pSYN FVIII 269 protein sequence (FVIII Fc with 72 AE-XTEN at amino acid 18) SEP ID NO: 115

1 MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQGAP

TSESATPESG

51 PGSEPATSGS ETPGTSESAT PESGPGSEPA TSGSETPGTS

ESATPESGPG

101 TSTEPSEGSA PGASSSDLGE LPVDARFPPR VPKSFPFNTS

VVYKKTLFVE

151 FTDHLFNIAK PRPPWMGLLG PTIQAEVYDT VVITLKNMAS

HPVSLHAVGV

201 SYWKASEGAE YDDQTSQREK EDDKVFPGGS HTYVWQVLKE

NGPMASDPLC

251 LTYSYLSHVD LVKDLNSGLI GALLVCREGS LAKEKTQTLH

KFILLFAVFD

301 EGKSWHSETK NSLMQDRDAA SARAWPKMHT VNGYVNRSLP

GLIGCHRKSV

351 YWHVIGMGTT PEVHSIFLEG HTFLVRNHRQ ASLEISPITF

LTAQTLLMDL

401 GQFLLFCHIS SHQHDGMEAY VKVDSCPEEP QLRMKNNEEA

EDYDDDLTDS

451 EMDVVRFDDD NSPSFIQIRS VAKKHPKTWV HYIAAEEEDW

DYAPLVLAPD

501 DRSYKSQYLN NGPQRIGRKY KKVRFMAYTD ETFKTREAIQ

HESGILGPLL 551 YGEVGDTLLI IFKNQASRPY NIYPHGITDV RPLYSRRLPK

GVKHLKDFPI

601 LPGEIFKYKW TVTVEDGPTK SDPRCLTRYY SSFVNMERDL

ASGLIGPLLI

651 CYKESVDQRG NQIMSDKRNV ILFSVFDENR SWYLTENIQR

FLPNPAGVQL

701 EDPEFQASNI MHS INGYVFD SLQLSVCLHE VAYWYILSIG

AQTDFLSVFF

751 SGYTFKHKMV YEDTLTLFPF SGETVFMSME NPGLWILGCH

NSDFRNRGMT

801 ALLKVSSCDK NTGDYYEDSY EDISAYLLSK NNAIEPRSFS

QNPPVLKRHQ

851 AEITRTTLQS DQEEIDYDDT I SVEMKKEDF DIYDEDENQS

PRSFQKKTRH

901 YFIAAVERLW DYGMSSSPHV LRNRAQSGSV PQFKKVVFQE

FTDGSFTQPL

951 YRGELNEHLG LLGPYIRAEV EDNIMVTFRN QASRPYSFYS

SLI SYEEDQR

1001 QGAEPRKNFV KPNETKTYFW KVQHHMAPTK DEFDCKAWAY

FSDVDLEKDV

1051 HSGLIGPLLV CHTNTLNPAH GRQVTVQEFA LFFTIFDETK

SWYFTENMER

1101 NCRAPCNIQM EDPTFKENYR FHAINGYIMD TLPGLVMAQD

QRIRWYLLSM

1151 GSNENIHSIH FSGHVFTVRK KEEYKMALYN LYPGVFETVE

MLPSKAGIWR

1201 VECLIGEHLH AGMSTLFLVY SNKCQTPLGM ASGHIRDFQI

TASGQYGQWA

1251 PKLARLHYSG SINAWSTKEP FSWIKVDLLA PMI IHGIKTQ

GARQKFSSLY

1301 ISQFIIMYSL DGKKWQTYRG NSTGTLMVFF GNVDSSGIKH

NIFNPPIIAR

1351 YIRLHPTHYS IRSTLRMELM GCDLNSCSMP LGMESKAISD

AQITASSYFT

1401 NMFATWSPSK ARLHLQGRSN AWRPQVNNPK EWLQVDFQKT

MKVTGVTTQG

1451 VKSLLTSMYV KEFLISSSQD GHQWTLFFQN GKVKVFQGNQ

DSFTPVVNSL

1501 DPPLLTRYLR IHPQSWVHQI ALRMEVLGCE AQDLYDKTHT

CPPCPAPELL

1551 GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF

NWYVDGVEVH

1601 NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN

KALPAPIEKT

1651 ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS

DIAVEWESNG

1701 QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC

SVMHEALHNH

1751 YTQKSLSLSP GK* pSYNFVIII 271 protein sequence (FVIII Fc with 42 AE-XTEN at amino acid 18) SEP ID NO: 116

1 MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQGAP

GSPAGSPTST

51 EEGTSESATP ESGPGSEPAT SGSETPASSS DLGELPVDAR

FPPRVPKSFP

101 FNTSVVYKKT LFVEFTDHLF NIAKPRPPWM GLLGPTIQAE

VYDTVVITLK

151 NMASHPVSLH AVGVSYWKAS EGAEYDDQTS QREKEDDKVF

PGGSHTYVWQ

201 VLKENGPMAS DPLCLTYSYL SHVDLVKDLN SGLIGALLVC

REGSLAKEKT

251 QTLHKFILLF AVFDEGKSWH SETKNSLMQD RDAASARAWP

KMHTVNGYVN

301 RSLPGLIGCH RKSVYWHVIG MGTTPEVHS I FLEGHTFLVR

NHRQASLEIS

351 PITFLTAQTL LMDLGQFLLF CHISSHQHDG MEAYVKVDSC

PEEPQLRMKN

401 NEEAEDYDDD LTDSEMDVVR FDDDNSPSFI QIRSVAKKHP

KTWVHYIAAE

451 EEDWDYAPLV LAPDDRSYKS QYLNNGPQRI GRKYKKVRFM

AYTDETFKTR

501 EAIQHESGIL GPLLYGEVGD TLLI IFKNQA SRPYNIYPHG

ITDVRPLYSR

551 RLPKGVKHLK DFPILPGEIF KYKWTVTVED GPTKSDPRCL

TRYYSSFVNM

601 ERDLASGLIG PLLICYKESV DQRGNQIMSD KRNVILFSVF

DENRSWYLTE

651 NIQRFLPNPA GVQLEDPEFQ ASNIMHSING YVFDSLQLSV

CLHEVAYWYI

701 LSIGAQTDFL SVFFSGYTFK HKMVYEDTLT LFPFSGETVF

MSMENPGLWI

751 LGCHNSDFRN RGMTALLKVS SCDKNTGDYY EDSYEDI SAY

LLSKNNAIEP

801 RSFSQNPPVL KRHQAEITRT TLQSDQEEID YDDTISVEMK

KEDFDIYDED

851 ENQSPRSFQK KTRHYFIAAV ERLWDYGMSS SPHVLRNRAQ

SGSVPQFKKV

901 VFQEFTDGSF TQPLYRGELN EHLGLLGPYI RAEVEDNIMV

TFRNQASRPY

951 SFYSSLISYE EDQRQGAEPR KNFVKPNETK TYFWKVQHHM

APTKDEFDCK

1001 AWAYFSDVDL EKDVHSGLIG PLLVCHTNTL NPAHGRQVTV

QEFALFFTIF

1051 DETKSWYFTE NMERNCRAPC NIQMEDPTFK ENYRFHAING

YIMDTLPGLV

1101 MAQDQRIRWY LLSMGSNENI HSIHFSGHVF TVRKKEEYKM

ALYNLYPGVF

1151 ETVEMLPSKA GIWRVECLIG EHLHAGMSTL FLVYSNKCQT

PLGMASGHIR 1201 DFQITASGQY GQWAPKLARL HYSGSINAWS TKEPFSWIKV

DLLAPMI IHG

1251 IKTQGARQKF SSLYISQFII MYSLDGKKWQ TYRGNSTGTL

MVFFGNVDSS

1301 GIKHNIFNPP I IARYIRLHP THYS IRSTLR MELMGCDLNS

CSMPLGMESK

1351 AISDAQITAS SYFTNMFATW SPSKARLHLQ GRSNAWRPQV

NNPKEWLQVD

1401 FQKTMKVTGV TTQGVKSLLT SMYVKEFLIS SSQDGHQWTL

FFQNGKVKVF

1451 QGNQDSFTPV VNSLDPPLLT RYLRIHPQSW VHQIALRMEV

LGCEAQDLYD

1501 KTHTCPPCPA PELLGGPSVF LFPPKPKDTL MISRTPEVTC

VVVDVSHEDP

1551 EVKFNWYVDG VEVHNAKTKP REEQYNSTYR VVSVLTVLHQ

DWLNGKEYKC

1601 KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSRDELTKN

QVSLTCLVKG

1651 FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT

VDKSRWQQGN

1701 VFSCSVMHEA LHNHYTQKSL SLSPGK* pSYN FVIII protein sequence 272 ( FVIII with 144 AE XTEN at amino acid 18 and 244 AE XTEN in B-domain- no Fc) SEP ID NO: 117

1 MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQGAP

TSESATPESG

51 PGSEPATSGS ETPGTSESAT PESGPGSEPA TSGSETPGTS

ESATPESGPG

101 TSTEPSEGSA PGSPAGSPTS TEEGTSESAT PESGPGSEPA

TSGSETPGTS

151 ESATPESGPG SPAGSPTSTE EGSPAGSPTS TEEGASSSDL

GELPVDARFP

201 PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL

LGPTIQAEVY

251 DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR

EKEDDKVFPG

301 GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG

LIGALLVCRE

351 GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD

AASARAWPKM

401 HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL

EGHTFLVRNH

451 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME

AYVKVDSCPE

501 EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI

RSVAKKHPKT

551 WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR

KYKKVRFMAY

601 TDETFKTREA IQHESGILGP LLYGEVGDTL LI IFKNQASR

PYNIYPHGIT 651 DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP

TKSDPRCLTR

701 YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR

NVILFSVFDE

751 NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHS INGYV

FDSLQLSVCL

801 HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF

PFSGETVFMS

851 MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED

SYEDI SAYLL

901 SKNNAIEPRS FSQNGAPGTS ESATPESGPG SEPATSGSET

PGTSESATPE

951 SGPGSEPATS GSETPGTSES ATPESGPGTS TEPSEGSAPG

SPAGSPTSTE

1001 EGTSESATPE SGPGSEPATS GSETPGTSES ATPESGPGSP

AGSPTSTEEG

1051 SPAGSPTSTE EGTSTEPSEG SAPGTSESAT PESGPGTSES

ATPESGPGTS

1101 ESATPESGPG SEPATSGSET PGSEPATSGS ETPGSPAGSP

TSTEEGTSTE

1151 PSEGSAPGTS TEPSEGSAPG SEPATSGSET PGTSESATPE

SGPGTSTEPS

1201 EGSAPASSPP VLKRHQAEIT RTTLQSDQEE IDYDDTISVE

MKKEDFDIYD

1251 EDENQSPRSF QKKTRHYFIA AVERLWDYGM SSSPHVLRNR

AQSGSVPQFK

1301 KVVFQEFTDG SFTQPLYRGE LNEHLGLLGP YIRAEVEDNI

MVTFRNQASR

1351 PYSFYSSLIS YEEDQRQGAE PRKNFVKPNE TKTYFWKVQH

HMAPTKDEFD

1401 CKAWAYFSDV DLEKDVHSGL IGPLLVCHTN TLNPAHGRQV

TVQEFALFFT

1451 IFDETKSWYF TENMERNCRA PCNIQMEDPT FKENYRFHAI

NGYIMDTLPG

1501 LVMAQDQRIR WYLLSMGSNE NIHSIHFSGH VFTVRKKEEY

KMALYNLYPG

1551 VFETVEMLPS KAGIWRVECL IGEHLHAGMS TLFLVYSNKC

QTPLGMASGH

1601 IRDFQITASG QYGQWAPKLA RLHYSGSINA WSTKEPFSWI

KVDLLAPMI I

1651 HGIKTQGARQ KFSSLYISQF I IMYSLDGKK WQTYRGNSTG

TLMVFFGNVD

1701 SSGIKHNIFN PPIIARYIRL HPTHYS IRST LRMELMGCDL

NSCSMPLGME

1751 SKAISDAQIT ASSYFTNMFA TWSPSKARLH LQGRSNAWRP

QVNNPKEWLQ

1801 VDFQKTMKVT GVTTQGVKSL LTSMYVKEFL ISSSQDGHQW

TLFFQNGKVK

1851 VFQGNQDSFT PVVNSLDPPL LTRYLRIHPQ SWVHQIALRM

EVLGCEAQDL

1901 Y* pSYN VWF 031 protein sequence ( VWF D1D2D'D3- 48aa long thrombin cleavable GS

linker-Fc) SEP ID NO: 118

1 MI PARFAGVL LALALILPGT LCAEGTRGRS STARCSLFGS DFVNTFDGSM

51 YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG

101 TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGFVARI

DGSGNFQVLL

151 SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL TSDPYDFANS

WALSSGEQWC

201 ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL

VDPEPFVALC

251 EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA

CSPVCPAGME

301 YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG

LCVESTECPC

351 VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV

TGQSHFKSFD

401 NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC

TRSVTVRLPG

451 LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL RIQHTVTASV

RLSYGEDLQM

501 DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG

LAEPRVEDFG

551 NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP

TFEACHRAVS

601 PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV

AWREPGRCEL

651 NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP

PGLYMDERGD

701 CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGFMHCTM

SGVPGSLLPD

751 AVLSSPLSHR SKRSLSCRPP MVKLVCPADN LRAEGLECTK

TCQNYDLECM

801 SMGCVSGCLC PPGMVRHENR CVALERCPCF HQGKEYAPGE

TVKIGCNTCV

851 CRDRKWNCTD HVCDATCSTI GMAHYLTFDG LKYLFPGECQ

YVLVQDYCGS

901 NPGTFRILVG NKGCSHPSVK CKKRVTILVE GGEIELFDGE

VNVKRPMKDE

951 THFEVVESGR YIILLLGKAL SVVWDRHLSI SVVLKQTYQE

KVCGLCGNFD

1001 GIQNNDLTSS NLQVEEDPVD FGNSWKVSSQ CADTRKVPLD

SSPATCHNNI

1051 MKQTMVDSSC RILTSDVFQD CNKLVDPEPY LDVCIYDTCS

CES IGDCAAF

1101 CDTIAAYAHV CAQHGKVVTW RTATLCPQSC EERNLRENGY

EAEWRYNSCA 1151 PACQVTCQHP EPLACPVQCV EGCHAHCPPG KILDELLQTC VDPEDCPVCE

1201 VAGRRFASGK KVTLNPSDPE HCQICHCDVV NLTCEACQEP

ISGGGGSGGG

1251 GSGGGGSGGG GSGGGGSGGG GSLVPRGSGG GGSGGGGSDK

THTCPPCPAP

1301 ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE

VKFNWYVDGV

1351 EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK

VSNKALPAPI

1401 EKTISKAKGQ PREPQVYTLP PSRDELTKNQ VSLTCLVKGF

YPSDIAVEWE

1451 SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV

FSCSVMHEAL

1501 HNHYTQKSLS LSPGK* pSYN VWF 034 protein sequence ( VWF D1D2D'D3- 288AE XTEN- 35aa long thrombin cleavable GS linker-Fc) SEP ID NO: 119

1 MI PARFAGVL LALALILPGT LCAEGTRGRS STARCSLFGS DFVNTFDGSM

51 YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG

101 TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGFVARI

DGSGNFQVLL

151 SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL TSDPYDFANS

WALSSGEQWC

201 ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL

VDPEPFVALC

251 EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA

CSPVCPAGME

301 YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG

LCVESTECPC

351 VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV

TGQSHFKSFD

401 NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC

TRSVTVRLPG

451 LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL RIQHTVTASV

RLSYGEDLQM

501 DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG

LAEPRVEDFG

551 NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP

TFEACHRAVS

601 PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV

AWREPGRCEL

651 NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP

PGLYMDERGD

701 CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGFMHCTM

SGVPGSLLPD

751 AVLSSPLSHR SKRSLSCRPP MVKLVCPADN LRAEGLECTK

TCQNYDLECM 801 SMGCVSGCLC PPGMVRHENR CVALERCPCF HQGKEYAPGE TVKIGCNTCV

851 CRDRKWNCTD HVCDATCSTI GMAHYLTFDG LKYLFPGECQ YVLVQDYCGS

901 NPGTFRILVG NKGCSHPSVK CKKRVTILVE GGEIELFDGE VNVKRPMKDE

951 THFEVVESGR YIILLLGKAL SVVWDRHLSI SVVLKQTYQE KVCGLCGNFD

1001 GIQNNDLTSS NLQVEEDPVD FGNSWKVSSQ CADTRKVPLD SSPATCHNNI

1051 MKQTMVDSSC RILTSDVFQD CNKLVDPEPY LDVCIYDTCS CES IGDCAAF

1101 CDTIAAYAHV CAQHGKVVTW RTATLCPQSC EERNLRENGY EAEWRYNSCA

1151 PACQVTCQHP EPLACPVQCV EGCHAHCPPG KILDELLQTC VDPEDCPVCE

1201 VAGRRFASGK KVTLNPSDPE HCQICHCDVV NLTCEACQEP ISGTSESATP

1251 ESGPGSEPAT SGSETPGTSE SATPESGPGS EPATSGSETP GTSESATPES

1301 GPGTSTEPSE GSAPGSPAGS PTSTEEGTSE SATPESGPGS EPATSGSETP

1351 GTSESATPES GPGSPAGSPT STEEGSPAGS PTSTEEGTST EPSEGSAPGT

1401 SESATPESGP GTSESATPES GPGTSESATP ESGPGSEPAT SGSETPGSEP

1451 ATSGSETPGS PAGSPTSTEE GTSTEPSEGS APGTSTEPSE GSAPGSEPAT

1501 SGSETPGTSE SATPESGPGT STEPSEGSAP DIGGGGGSGG GGSLVPRGSG

1551 GDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMI SRTPEV

TCVVVDVSHE

1601 DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY

1651 KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV

1701 KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK

LTVDKSRWQQ

1751 GNVFSCSVMH EALHNHYTQK SLSLSPGK* pSYN VWF 036 protein sequence ( VWF DlD2D'D-98aa long thrombin cleavable GS

linker-Fc) SEP ID NO: 120

1 MI PARFAGVL LALALILPGT LCAEGTRGRS STARCSLFGS DFVNTFDGSM

51 YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG

101 TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGFVARI DGSGNFQVLL

151 SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL TSDPYDFANS WALSSGEQWC 201 ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL

VDPEPFVALC

251 EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA

CSPVCPAGME

301 YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG

LCVESTECPC

351 VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV

TGQSHFKSFD

401 NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC

TRSVTVRLPG

451 LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL RIQHTVTASV

RLSYGEDLQM

501 DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG

LAEPRVEDFG

551 NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP

TFEACHRAVS

601 PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV

AWREPGRCEL

651 NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP

PGLYMDERGD

701 CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGFMHCTM

SGVPGSLLPD

751 AVLSSPLSHR SKRSLSCRPP MVKLVCPADN LRAEGLECTK

TCQNYDLECM

801 SMGCVSGCLC PPGMVRHENR CVALERCPCF HQGKEYAPGE

TVKIGCNTCV

851 CRDRKWNCTD HVCDATCSTI GMAHYLTFDG LKYLFPGECQ

YVLVQDYCGS

901 NPGTFRILVG NKGCSHPSVK CKKRVTILVE GGEIELFDGE

VNVKRPMKDE

951 THFEVVESGR YIILLLGKAL SVVWDRHLSI SVVLKQTYQE

KVCGLCGNFD

1001 GIQNNDLTSS NLQVEEDPVD FGNSWKVSSQ CADTRKVPLD

SSPATCHNNI

1051 MKQTMVDSSC RILTSDVFQD CNKLVDPEPY LDVCIYDTCS

CES IGDCAAF

1101 CDTIAAYAHV CAQHGKVVTW RTATLCPQSC EERNLRENGY

EAEWRYNSCA

1151 PACQVTCQHP EPLACPVQCV EGCHAHCPPG KILDELLQTC

VDPEDCPVCE

1201 VAGRRFASGK KVTLNPSDPE HCQICHCDVV NLTCEACQEP

ISGGGGSGGG

1251 GSGGGGSGGG GSGGGGSGGG GSGGGGSGGG GSGGGGSGGG

GSGGGGSGGG

1301 GSGGGGSGGG GSGGGGSGGG GSLVPRGSGG GGSGGGGSDK

THTCPPCPAP

1351 ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE

VKFNWYVDGV

1401 EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK

VSNKALPAPI 1451 EKTISKAKGQ PREPQVYTLP PSRDELTKNQ VSLTCLVKGF

YPSDIAVEWE

1501 SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV

FSCSVMHEAL

1551 HNHYTQKSLS LSPGK* pSYN Fc-015 protein sequence ( IgG-Fc domain) SEP ID NO: 121

1 METDTLLLWV LLLWVPGSTG DKTHTCPPCP APELLGGPSV FLFPPKPKDT

51 LMI SRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK

PREEQYNSTY

101 RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK

GQPREPQVYT

151 LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN

YKTTPPVLDS

201 DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS

LSLSPGK*

Example 15: FVIII-XTEN-Fc:VWF-Fc heterodimers have maintained normal FVIII specific activity as compared to wild type BDD-FVIII.

[0383] The FVIII specific activity of FVIII-XTEN-Fc: VWF-Fc heterodimers were determined. Heterodimers were purified using a two-step chromatography process. A weak anion exchange resin was used, followed by affinity chromatography. The final purified product had acceptable purity by SEC-HP LC. The specific activity was compared to B-domain deleted FVIII (BDD-FVIII), as measured by FVIII chromogenic assay and A280 concentration. The data are presented in Table 26. All tested molecules had demonstrated comparable FVIII specific activities to BDD-FVIII. Purity and the presence of each moiety of the molecules were confirmed by SDS-PAGE and western blotting.

Table 26: FVIII specific activity of FVIII-XTEN-Fc: VWF-Fc heterodimers

[0384] The half-lives of rFVIII-XTEN/D'D3 and BDD-FVIII were compared in

HemA Mice (Figure 15; Table 27). As Figure 15 shows, rFVIII-XTEN/D'D3 achieved a half-life that was four fold longer than the half-life achieved by BDD-

FVIII.

Table 27: rFVIII-XTEN/D'D3 and BDD-FVIII in HemA mice

Example 16: FVIII-XTEN-Fc:VWF-Fc heterodimer's potency (FVIII activity) in hemostasis as measured by one stage aPTT assay

[0385] The potency of FVIII -XTEN-Fc: VWF-Fc heterodimers in hemostasis was evaluated by their FVIII specific aPTT activity as summarized in Table 28. As demonstrated by Table 28, while the addition of the VWF D'D3 fragment and the insertion of XTEN into the intra-domains of FVIII reduce the FVIII specific aPTT activity of the heterodimers (as indicated by the FVIII155/VWF031 data and the FVIII205/VWF031 data), XTEN insertions in the FVIII B domain region or C- terminus of the VWF D'D3 fragment have no negative effect on the FVIII specific aPTT activity (as indicated by the FVIII 169/VWF031 data and the

FVIII 169/VWF034 data). Compared to dual-chain BDD-FVIII (dcBDD-FVIII), FVIII155/VWF031, FVIII 169/VWF031, FVIII 169/VWF034 and

VWF205/VWF031 showed reduction of specific aPTT activity by 2.5-fold, 2.8- fold, 2.6-fold and 5.5-fold, respectively.

Table 28: FVIII specific aPTT activity of FVIII-XTEN-Fc:VWF-Fc heterodimers

FVIII specific aPTT assay

[0386] FVIII variants were diluted with aPTT buffer (0.15 M NaCl, 0.05 M Tris-

HC1, 1% BSA, pH 7.4) to the linear assay range (200- 1.6 mU/mL). 50 μΐ, of diluted samples or standards were sequentially mixed with 50 of 37°C na^'ive human HemA pooled plasma, 50 μΐ, of 37°C aPTT reagent (ACTIN® FSL activated cephaloplastin reagent - Dade Behring, reference # B4219-2) and incubated at 37 °C for 4 minutes. 50 μΐ of 20 mM CaCl₂ (Dade Behring [reference # ORF037]) was then added to the reaction mixture to start the clotting reactions. Using the clotting time of each sample (the length of time from the addition of CaCl₂ until the onset of clot formation), the aPTT activity was calculated against the standard that was generated with the 8^th international standard FVIII concentrate. Specific aPTT activity was calculated against the protein

concentration of each molecule that measured by OD280.

Example 17: In vivo efficacy of FVIII-XTEN-Fc:VWF-Fc heterodimer in HemA mice Tail Clip bleeding model

[0387] To further access the hemostasis potency of the heterodimers, the acute efficacy of FVIII 169/VWF034 and FVIII205/VWF031 was evaluated in comparison with BDD-FVIII in the HemA mice Tail clip bleeding model. HemA mice were treated with a single IV injection of BDD-FVIII at 200, 65 and 20 IU/kg to generate the post tail clip injury blood loss control level. Blood loss from mice treated with 200 IU/kg of FVIII 169/VWF034 or FVIII205/VWF031 was compared to that of the BDD-FVIII treated control group mice to estimate their potency on hemostasis. Vehicle treated animals were used to generate blood loss baseline for the model. As shown in Figure 16, significant reduction in blood loss was observed from all FVIII treatment groups compared to that of the vehicle treated animals (p<0.05). Both FVIII 169/VWF034 and FVIII205/VWF031 are efficacious in the HemA mice Tail Clip model. Compared to BDD-FVIII, about 3 fold lower potency was observed for FVIII 169/VWF034, as demonstrated by the similar blood loss reduction achieved by 65 IU/kg BDD-FVIII and 200 IU/kg FVIII169/VWF034. As for FVIII205/VWF034, a 10 fold potency reduction has been observed, as demonstrated by the similar blood loss reduction achieved by 20 IU/kg BDD-FVIII and 200 IU/kg FVIII205/VWF031. [0388] Even though FVIII69/VWF034 and FVIII205/VWF031 had similar specific FVIII chromogenic activity compared to rBDD-FVIII, their FVIII aPTT activity and in vivo potency were both reduced due to the modifications of the molecules. Those data indicate that the aPTT activity of a FVIII molecule is a more accurate measurement on predicating its in vivo potency on hemostasis than the FVIII chromogenic activity.

HemA mice Tail clip bleeding model

[0389] 8-10 weeks old male HemA mice were used for the study. Prior to tail clip injury, mice were anesthetized with a 50 mg/kg Ketamine/0.5 mg/kg

Dexmedetomidine cocktail and placed on a 37°C heating pad to help maintain the body temperature. The tails of the mice were then be immersed in 37°C water for 10 minutes to dilate the lateral vein. After vein dilation, rFVIII or vehicle solution were injected via the tail vein and 5 min later, the distal 1 cm of the tail was cut off using a #11 scalpel with straight edge. The shed blood was collected into 13 ml of 37°C warm saline for 30 minutes and the mice were then euthanized while still under anesthesia by bilateral thoracotomy. Blood loss was quantified gravimetrically by weight change of the blood collection tubes before and after blood was collected in gram, which translated into milliliter (mL) of blood loss volume (lg weight change = 1 mL blood loss).

[0390] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0391] Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

All patents and publications cited herein are incorporated by reference herein in their entirety.

Claims

WHAT IS CLAIMED IS:

1. A chimeric protein comprising (i) a von Willebrand Factor (VWF) protein comprising the D' domain and the D3 domain of VWF, (ii) an XTEN sequence, and (iii) a FVIII protein, wherein the VWF fragment and the XTEN sequence are linked by an optional linker, wherein the VWF fragment or the XTEN sequence is linked to or associated with the FVIII protein.

2. A chimeric protein comprising a formula, which comprises:

(a) V-X-FVIII,

(b) FVIII-X-V,

(c) V-X:FVIII,

(d) X-V:FVIII,

(e) FVIII:V-X, or

(f) FVIII:X-V,

wherein V comprises a VWF fragment,

X comprises one or more XTEN sequences,

FVIII comprises a FVIII protein;

(-) is a peptide bond or one or more amino acids; and

(:) is a covalent bond or a non-covalent bond.

3. The chimeric protein of claim 1 or 2, wherein the XTEN sequence is linked to the FVIII protein by a linker.

4. The chimeric protein of claim 3, wherein the linker is a cleavable linker.

5. The chimeric protein of any one of claims 1 to 4, which comprises a single polypeptide chain comprising the VWF fragment, the XTEN sequence and the FVIII protein.

6. The chimeric protein of any one of claims 1 to 4, which comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises FVIII and the second polypeptide chain comprises the VWF fragment and the XTEN sequence.

7. The chimeric protein of any one of claims 1 to 6, further comprising (iv) an Ig constant region or a portion thereof linked to either the VWF fragment, the XTEN sequence, or the FVIII protein, or any combination thereof.

8. A chimeric protein comprising a formula, which comprises:

(g) V-L2-X-L1-F1 :FVm-L3-F2;

GO V-L2-X-L1-F1 :F2-L3-FVIII;

(i) F1-L1-X-L2-V: FVIII-L3-F2;

0⁾ F 1 -L 1 -X-L2-V:F2-L3 -FVIII;

(k) V-L2-X-L 1 -F 1 -L4-FVIII-L3 -F2;

(1) F2-L3-FVIII-L4-F 1 -L 1 -X-L2-V;

(m) FVIII-L3-F2-L4-V-L2-X-L1-F1; and

(n) F 1 -L 1 -X-L2-V-L4-F2-L3-FVIII,

wherein V comprises a VWF fragment,

each of LI, L2, and L3 comprises an optional linker,

L4 is an optional linker,

FVIII comprises a FVIII protein,

X comprises one or more XTEN sequences,

Fl comprises an optional Ig constant region or a portion thereof,

F2 comprises an optional additional Ig constant region or a portion thereof;

(-) is a peptide bond or one or more amino acids; and

(:) is a covalent bond or a non-covalent bond.

9. The chimeric protein of any one of claims 7 or 8, wherein the Ig constant region or a portion thereof extends a half-life of the VWF fragment.

10. The chimeric protein of any one of claims 7 to 9, wherein the Ig constant region or a portion thereof comprises a first Fc region, which is linked to the XTEN sequence or the VWF fragment.

11. The chimeric protein of claim 10, wherein the Ig constant region or a portion thereof is linked to the XTEN sequence by a linker.

12. The chimeric protein of claim 12, wherein the linker comprises a cleavable linker.

13. The chimeric protein of any one of claims 7 to 12, further comprising an additional Ig constant region or a portion thereof.

14. The chimeric protein of claim 13, wherein the additional Ig constant region or a portion thereof comprises an additional Fc region.

15. The chimeric protein of claim 14, wherein the additional Ig constant region or a portion thereof extends the half-life of a FVIII protein.

16. The chimeric protein of any one of claims 13 to 15, wherein the additional Ig constant region or a portion thereof is linked to the FVIII protein.

17. The chimeric protein of claim 16, wherein the second Fc region is further linked to the VWF fragment by a linker.

18. The chimeric protein of claim 17, wherein the linker is a processable linker.

19. The chimeric protein of any one of claims 8 to 18, wherein L4 is a processable linker.

20. The chimeric protein of any one of claims 7 to 19, wherein the additional Ig constant region or a portion thereof is associated with the Ig constant region or a portion thereof.

21. The chimeric protein of claim 20, wherein the additional Ig constant region or a portion thereof is associated with the Ig constant region or a portion thereof by a covalent bond.

22. The chimeric protein of claim 21, wherein the covalent bond is a disulfide bond.

23. The chimeric protein of any one of claims 1 to 22, wherein the VWF fragment is associated with the FVIII protein by a non-covalent bond.

24. The chimeric protein of any one of claims 1 to 23, wherein the half-life of the FVIII protein, is extended compared to a FVIII protein without the VWF fragment or compared to wild type FVIII.

25. The chimeric protein of claim 24, wherein the half-life of the FVIII is extended at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, or at least about 12 times longer than a FVIII protein without the VWF fragment or compared to wild type FVIII..

26. The chimeric protein of claim 24, wherein the half-life of the Factor VIII is at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, at least about 28 hours, at least about 29 hours, at least about 30 hours, at least about 31 hours, at least about 32 hours, at least about 33 hours, at least about 34 hours, at least about 35 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours.

27. A chimeric protein comprising (i) a FVIII protein, (ii) an XTEN sequence, and (iii) an Ig constant region or a portion thereof, wherein the XTEN sequence is linked to the FVIII protein by an optional linker at the N-terminus or C terminus of the FVIII protein or is inserted between two amino acids of at least one or more insertion sites in the FVIII protein and wherein the Ig constant region or a portion thereof is linked to or associated with the FVIII protein or the XTEN sequence.

28. The chimeric protein of claim 27, wherein the XTEN sequence and the Ig constant region or a portion thereof extends the half-life of the FVIII protein.

29. The chimeric protein of claim 27 or 28, wherein the VWF binding site is located in the A3 domain or the C2 domain of the FVIII protein or both the A3 domain and the C2 domain.

30. The chimeric protein of claim 29, wherein the VWF binding site comprises the amino acid sequence corresponding to amino acids 1669 to 1689 and 2303 to 2332 of SEQ ID NO: 4.

31. The chimeric protein of any one of claims 27 to 30, wherein the half-life of the FVIII protein is extended compared to a FVIII protein without the Ig constant region or a portion thereof or a FVIII protein without the XTEN sequence.

32. The chimeric protein of claim 31, wherein the half-life of the FVIII protein is extended at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, or at least about 12 times longer than a FVIII protein without the Ig constant region or a portion thereof or a FVIII protein without the XTEN sequence or wild-type FVIII.

33. The chimeric protein of claim 31, wherein the half-life of the FVIII protein is at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours.

34. The chimeric protein of any one of claims 27 to 33, wherein the Ig constant region or a portion thereof comprises a first Fc region.

35. The chimeric protein of any one of claims 27 to 34, further comprising an additional Ig constant region or a portion thereof.

36. The chimeric protein of claim 35, wherein the additional Ig constant region or a portion thereof comprises a second Fc region, which is linked to or associated with the first Fc region.

37. The chimeric protein of claim 36, wherein the second Fc region is associated with the first Fc region by a covalent bond.

38. The chimeric protein of claim 36, wherein the first Fc region is linked to the second Fc region by a linker.

39. The chimeric protein of claim 38, wherein the linker is a processable linker.

40. The chimeric protein of any one of claims 27 to 34, which comprises a single polypeptide chain comprising the FVIII protein, the XTEN sequence, and the Ig constant region or a portion thereof.

41. The chimeric protein of any one of claims 27 to 34, which comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a heavy chain of the FVIII protein, the second polypeptide chain comprises a light chain of the FVIII protein and the XTEN sequence and the Ig constant region or a portion thereof.

42. The chimeric protein of any one of claims 35 to 39, which comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises the FVIII protein, the XTEN sequence, and the Ig constant region and the second polypeptide chain comprises the additional Ig constant region or a portion thereof.

43. The chimeric protein of any one of claims 35 to 39, which comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first polypeptide chain comprises a heavy chain of the FVIII protein and the XTEN sequence, the second polypeptide chain comprises a light chain of the FVIII protein and the Ig constant region or a portion thereof, and the third polypeptide chain comprises the additional Ig constant region or a portion thereof.

44. The chimeric protein of any one of claims 35 to 39, which comprises a single chain polypeptide comprising the FVIII protein, the XTEN sequence, the Ig constant region or a portion thereof, and the additional Ig constant region or a portion thereof.

45. A chimeric protein comprising (i) a FVIII protein, (ii) an XTEN sequence, (iii) a VWF fragment, and (iv) an Ig constant region or a portion thereof, which comprises the D' domain and the D3 domain of VWF, wherein the XTEN sequence is linked to the FVIII protein by an optional linker at the N-terminus or C terminus of the FVIII protein or inserted immediately downstream of one or more insertion sites in the FVIII protein, the VWF fragment is linked to or associated with the FVIII protein or the XTEN sequence, and the Ig constant region or a portion thereof is linked to the FVIII protein, the XTEN sequence, the VWF fragment, or any combinations thereof.

46. A chimeric protein comprising a formula, which comprises:

( 1 ) F VIII(X 1 )-L 1 -F 1 : V-L2-X2-L3 -F2;

(2) FVIII(X1)-L1-F1 :F2-L3-X2-L2-V;

(3) F 1 -L 1 -FVIII(X 1 ) : V-L2-X2-L3 -F2;

(4) F 1 -L 1 -FVIII(X1 );F2-L3-X2-L2-V;

(5) FVIII(X1 )-L 1 -F 1 -L4-V-L2-X2-L3-F2;

(6) FVIII(X1 )-L 1 -F 1 -L4-F2-L3-X2-L2-V;

(7) F1-L1-FVIII(X1)-L4-V-L2-X2-L3-F2, or

(8) F 1 -L 1 -FVIII(X1 )-L4-F2-L3-X2-L2-V,

wherein FVIII(Xl) comprises a FVIII protein and an XTEN sequence, wherein the

XTEN sequence is linked to the N-terminus or C-terminus of the FVIII protein or inserted immediately downstream of one or more amino acids ("one or more insertion site") in the FVIII protein;

each of LI, L2, or L3 comprises an optional linker;

L4 is a linker;

X2 comprises one or more XTEN sequences;

Fl comprises an Ig constant region or a portion thereof;

V comprises a VWF fragment;

(-) is a peptide bond or one or more amino acids; and

(:) comprises a covalent bond or a non-covalent bond.

47. The chimeric protein of claim 45 or 46, wherein the VWF fragment does not bind to a VWF clearance receptor.

48. The chimeric protein of any one of claims 45 to 47, wherein the VWF fragment is capable of protecting the FVIII protein from cleavage by one or more protease, protecting the FVIII protein from activation, stabilizing the heavy chain and/or the light chain of the FVIII protein, or preventing clearance of the FVIII protein by one or more scavenger receptors.

49. The chimeric protein of any one of claims 45 to 48, wherein the Ig constant region or a portion thereof inhibits or prevents endogenous VWF from binding to the FVIII protein by shielding or blocking a VWF binding site on the FVIII protein.

50. The chimeric protein of claim 49, wherein the VWF binding site is located in the A3 domain or the C2 domain of the FVIII protein or both the A3 domain and the C2 domain.

51. The chimeric protein of claim 49 or 50, wherein the VWF binding site comprises the amino acid sequence corresponding to amino acids 1669 to 1689 and 2303 to 2332 of SEQ ID NO: 4.

52. The chimeric protein of any one of claims 45 to 51, wherein a half-life of the FVIII protein is extended compared to a FVIII protein without the VWF fragment.

53. The chimeric protein of claim 52, wherein the half-life of the FVIII protein is extended at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, or at least about 12 times longer than wild type FVIII.

54. The chimeric protein of claim 52, wherein the half-life of the FVIII protein is at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours.

55. The chimeric protein of any one of claims 45 to 54, which comprises a single polypeptide chain comprising the FVIII protein, the XTEN sequence, the VWF fragment, and the Ig constant region or a portion thereof.

56. The chimeric protein of any one of claims 45 to 54, wherein the Ig constant region or a portion thereof comprises a first Fc region.

57. The chimeric protein of any one of claims 45 to 54, wherein the Ig constant region or a portion thereof is linked to the VWF fragment by an optional linker.

58. The chimeric protein of claim 57, wherein the linker comprises a cleavable linker.

59. The chimeric protein of claim 45 to 50, which further comprises an additional Ig constant region or a portion thereof, which is linked to the FVIII protein, the Ig constant region or a portion thereof, the VWF fragment, or any combinations thereof by an optional linker.

60. The chimeric protein of claim 59, wherein the additional Ig constant region or a portion thereof is linked to the FVIII protein by an optional linker.

61. The chimeric protein of claim 59 or 60, wherein the Ig constant region or a portion thereof is a second Fc region.

62. The chimeric protein of any one of claims 8 to 26, 42 to 44, and 46 to 61, wherein the Ig constant region or a portion thereof and the additional Ig constant region or a portion thereof are identical or different.

63. The chimeric protein of any one of claims 46 to 62, which comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises the FVIII protein, the XTEN sequence, and the Ig constant region or a portion thereof and the second polypeptide chain comprises the VWF fragment and the additional Ig constant region or a portion thereof.

64. The chimeric protein of any one of claims 46 to 62, which comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first polypeptide chain comprises a heavy chain of the FVIII protein and the XTEN sequence, the second polypeptide chain comprises a light chain of the FVIII protein and the Ig constant region or a portion thereof, and the third polypeptide chain comprises the VWF fragment and the additional Ig constant region or a portion thereof.

65. The chimeric protein of any one of claims 46 to 62, which comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first polypeptide chain comprises a heavy chain of the FVIII protein, the second polypeptide chain comprises a light chain of the FVIII protein, the XTEN sequence, and the Ig constant region or a portion thereof, and the third polypeptide chain comprises the VWF fragment and the additional Ig constant region or a portion thereof.

66. The chimeric protein of any one of claims 46 to 62, wherein a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a heavy chain of the FVIII protein and the XTEN sequence and the second polypeptide chain comprises a light chain of the FVIII protein, the Ig constant region or a portion thereof, the VWF fragment, and the additional Ig constant region or a portion thereof.

67. The chimeric protein of any one of claims 63 to 66, which further comprises an additional XTEN sequence linked to the VWF fragment or to the additional Ig constant region or a portion thereof.

68. The chimeric protein of any one of claims 1 to 26, wherein the FVIII protein is linked to an XTEN sequence at the C-terminus or the N-terminus of the FVIII protein or inserted immediately downstream of one or more amino acids in the FVIII protein or any combinations thereof.

69. The chimeric protein of any one of claims 27 to 68, wherein the FVIII protein is linked to at least two XTEN sequences, at least three XTEN sequences, at least four XTEN sequences, at least five XTEN sequences, or at least six XTEN sequences.

70. The chimeric protein of any one of claims 1 to 69, wherein the FVIII protein comprises one or more domains of FVIII selected from the group consisting of an A 1 domain, al acidic region, an A2 domain, a2 acidic region, a B domain, an A3 domain, a3 acidic region, a CI domain, a C2 domain, one or more fragments thereof, and any combinations thereof.

71. The chimeric protein of any one of claims 27 to 70, wherein the one or more insertion sites in the FVIII protein is located within one or more domains of the FVIII protein selected from the group consisting of the Al domain, the al acidic region, the A2 domain, the a2 acidic region, the A3 domain, the B domain, the CI domain, the C2 domain, and any combinations thereof or between one or more domains of the FVIII protein selected from the group consisting of the Al domain and al acidic region, the al acidic region and A2 domain, the A2 domain and a2 acidic region, the a2 acidic region and B domain, the B domain and A3 domain, the A3 domain and CI domain, the CI domain and C2 domain, and any combinations thereof or between two domains of the FVIII protein selected from the group consisting of the Al domain and al acidic region, the al acidic region and A2 domain, the A2 domain and a2 acidic region, the a2 acidic region and B domain, the B domain and A3 domain, the A3 domain and CI domain, the CI domain and C2 domain, and any combinations thereof.

72. The chimeric protein of any one of claims 27 to 71 , wherein the one or more insertion sites in the FVIII protein are one or more amino acids selected from the group consisting of the amino acid residues in Table 7, Table 8, Table 9 and Table 10.

73. The chimeric protein of claim 72, wherein the XTEN sequence inserted at the insertion site corresponding to amino acids 3R of SEQ ID NO: 4 further comprises an amino acid sequence of ATR.

74. The chimeric protein of any one of claims 27 to 71 , wherein the one or more insertion sites in the FVIII protein are located immediately downstream of one or more amino acids selected from the group consisting of:

(1) amino acid 3, (2) amino acid 18, (3) amino acid 22,

(4) amino acid 26, (5) amino acid 32, (6) amino acid 40,

(7) amino acid 60, (8) amino acid 65, (9) amino acid 81 ,

(10) amino acid 116, (11) amino acid 119, (12) amino acid 130,

(13) amino acid 188, (14) amino acid 211, (15) amino acid 216,

(16) amino acid 220, (17) amino acid 224, (18) amino acid 230,

(19) amino acid 333, (20) amino acid 336, (21) amino acid 339,

(22) amino acid 375, (23) amino acid 399, (24) amino acid 403,

(25) amino acid 409, (26) amino acid 416, (26) amino acid 442,

(28) amino acid 487, (29) amino acid 490, (30) amino acid 494,

(31) amino acid 500, (32) amino acid 518, (33) amino acid 599,

(34) amino acid 603, (35) amino acid 713, (36) amino acid 745,

(37) amino acid 1656, (38) amino acid 1711, (39) amino acid 1720,

(40) amino acid 1725, (41) amino acid 1749, (42) amino acid 1796,

(43) amino acid 1802, (44) amino acid 1827, (45) amino acid 1861,

(46) amino acid 1896, (47) amino acid 1900, (48) amino acid 1904, (49) amino acid 1905, (50) amino acid 1910, (51) amino acid 1937,

(52) amino acid 2019, (53) amino acid 2068, (54) amino acid 2111,

(55) amino acid 2120, (56) amino acid 2171 , (57) amino acid 2188,

(58) amino acid 2227, (59) amino acid 2277, and

(60) two or more combinations thereof.

75. The chimeric protein of any one of claims 1 to 74, wherein the FVIII protein comprises B domain or a portion thereof.

76. The chimeric protein of claim 75, wherein the FVIII protein is SQ B domain deleted

FVIII.

77. The chimeric protein of any one of claims 1 to 76, wherein the FVIII protein comprises single chain FVIII.

78. The chimeric protein of claim 77, wherein the single chain FVIII contains at least one amino acid substitution at a residue corresponding to residue 1648, residue 1645, or both of full-length mature Factor VIII polypeptide (SEQ ID NO: 4) or residue 754, residue 751, or both of SQ BDD Factor VIII (SEQ ID NO: 6).

79. The chimeric protein of claim 78, wherein the amino acid substitution is an amino acid other than arginine.

80. The chimeric protein of any one of claims 1 to 76, wherein the FVIII protein comprises a heavy chain of FVIII and a light chain of Factor VIII, wherein the heavy chain and the light chain are associated with each other by a metal bond.

81. The chimeric protein of any one of claims 1 to 80, wherein the FVIII protein has a low affinity to or does not bind to a low-density lipoprotein receptor-related protein (LRP).

82. The chimeric protein of claim 81 , wherein the FVIII protein contains at least one amino acid substitution that lowers the affinity to or eliminates the binding to the LRP.

83. The chimeric protein of claim 82, wherein the at least one amino acid substitution is at a residue corresponding to residue 471, residue 484, residue 487, residue 490, residue 497, residue 2092, residue 2093 or two or more combinations thereof of full-length mature FVIII.

84. The chimeric protein of claim 83, wherein the amino acid substitution at residue 471, 484, or 497 is an amino acid other than Arginine, the amino acid substitution at residue 487 is an amino acid other than Tyrosine, the amino acid substitution at residue 2092 is an amino acid other than Lysine, or the amino acid substitution at residue 2093 is an amino acid other than phenylalanine.

85. The chimeric protein of any one of claims 1 to 84, wherein the FVIII protein contains at least one amino acid substitution, which induces the FVIII protein to be more stable than a FVIII protein without the substitution.

86. The chimeric protein of claim 85, wherein the A2 domain and the A3 domain of the FVIII protein are associated to each other by a covalent bond.

87. The chimeric protein of claim 85 or 86, wherein the at least one amino acid substitution is at a residue corresponding to residue 664, residue 1826, residue 662, or residue 1828 of full-length mature FVIII, or two or more combinations thereof.

88. The chimeric protein of claim 87, wherein the FVIII protein contains (a) cysteine at the residue corresponding to residue 664 of full-length mature FVIII and cysteine at the residue corresponding to residue 1826 of full-length mature FVIII or (b) cysteine at the residue corresponding to residue 662 of full-length mature FVIII and cysteine at the residue corresponding to residue 1828 of full-length mature FVIII.

89. The chimeric protein of any one of claims 1 to 26 and 45 to 88, wherein the VWF fragment is not amino acids 764 to 1274 of SEQ ID NO: 2.

90. The chimeric protein of any one of claims 1 to 26 and 45 to 89, wherein the amino acid sequence of the D^* domain is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764 to 866 of SEQ ID NO: 2.

91. The chimeric protein of any one of claims 1 to 26 and 45 to 90, wherein the amino acid sequence of the D3 domain is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 867 to 1240 of SEQ ID NO: 2.

92. The chimeric protein of any one of claims 1 to 26 and 45 to 91 , wherein the VWF fragment is a monomer.

93. The chimeric protein of any one of claims 1 to 26 and 45 to 91, wherein the VWF fragment comprises at least two VWF fragments, at least three VWF fragments, at least four VWF fragments, at least five VWF fragments, or at least six VWF fragments.

94. The chimeric protein of any one of claims 1 to 26 and 45 to 93, wherein the VWF fragment comprises an amino acid at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764 to 1240 of SEQ ID NO: 2.

95. The chimeric protein of claim 94, wherein the VWF fragment consists essentially of or consists of amino acids 764 to 1240 of SEQ ID NO: 2.

96. The chimeric protein of any one of claims 1 to 26 and 45 to 95, wherein the VWF fragment contains at least one amino acid substitution at a residue corresponding to residue 1099, residue 1142, or both residues 1099 and 1142 of SEQ ID NO: 2.

97. The chimeric protein of claim 96, wherein the VWF fragment contains an amino acid other than cysteine substituted for a residue corresponding to residue 1099, residue 1142, or both residues 1099 and 1142 of SEQ ID NO: 2.

98. The chimeric protein of any one of claims 1 to 26 and 45 to 97, wherein the VWF fragment further comprises the Dl domain, the D2 domain, or the Dl and D2 domains of VWF.

99. The chimeric protein of any one of claims 1 to 27 and 45 to 98, wherein the VWF fragment further comprises a VWF domain selected from the group consisting of the Al domain, the A2 domain, the A3 domain, the D4 domain, the Bl domain, the B2 domain, the B3 domain, the CI domain, the C2 domain, the CK domain, one or more fragments thereof, and any combinations thereof.

100. The chimeric protein of any one of claims 1 to 27 and 45 to 99, wherein the VWF fragment consists essentially of or consists of: (1) the D' and D3 domains of VWF or fragments thereof; (2) the Dl, D', and D3 domains of VWF or fragments thereof; (3) the D2, D', and D3 domains of VWF or fragments thereof; (4) the Dl, D2, D', and D3 domains of VWF or fragments thereof; or (5) the Dl, D2, D', D3, and Al domains of VWF or fragments thereof.

101. The chimeric protein of any one of claims 1 to 27 and 45 to 100, wherein the VWF fragment further comprises a signal peptide of VWF or FVIII which is operably linked to the VWF fragment.

102. The chimeric protein of any one of claims 1 to 101, wherein one or more of the linkers have a length of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, or 2000 amino acids.

103. The chimeric protein of any one of claims 1 to 101, wherein one or more of the linkers have a length of about 1 to about 2000 amino acids.

104. The chimeric protein of claim 103, wherein one or more of the linkers have a length of at least about 20, 35, 42, 48, 73, 75, 95, 98, 144, 288, 324, 333, 576, or 864 amino acids.

105. The chimeric protein of any one of claims 1 to 104, wherein one or more of the linkers comprise a Gly/Ser peptide.

106. The chimeric protein of claim 105, wherein the Gly/Ser peptide has a formula of (Gly₄Ser)_n (SEQ ID NO: 139) or Ser(Gly₄Ser)_n (SEQ ID NO: 140), wherein n is a positive integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

107. The chimeric protein of claim 106, wherein the (Gly₄Ser)_n linker is (Gly₄Ser)₃ (SEQ ID NO: 63) or (Gly₄Ser)₄ (SEQ ID NO: 138).

108. The chimeric protein of any one of claims 1 to 107, wherein the XTEN sequence is selected from the group consisting of AE42, AE72, AE864, AE576, AE288, AE144, AG864, AG576, AG288, and AG144.

109. The chimeric protein of claim 108, wherein the XTEN sequence is selected from the group consisting of SEQ ID NO: 36; SEQ ID NO: 37; SEQ ID NO: 38; SEQ ID NO: 39; SEQ ID NO: 40; SEQ ID NO: 41; SEQ ID NO: 42; SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 127.

110. The chimeric protein of claim 109, wherein the XTEN sequence is AE288 or AG288.

111. The chimeric protein of any one of claims 1 to 110, wherein the linker comprises at least one first cleavage site at the N-terminus of the linker, at least one second cleavage site at the C-terminus of the linker, or both.

112. The chimeric protein of any one of claims 1 to 111, wherein the linker comprises 20 amino acids, 35 amino acids, 48 amino acids, 73 amino acids, or 95 amino acids.

113. The chimeric protein of claim 112, wherein the linker is 48 amino acids thrombin cleavage linker.

114. The chimeric protein of claim 112, wherein the linker is 35 amino acids thrombin cleavage linker.

115. The chimeric protein of claim 111, wherein the one or more of the cleavage sites is TLDPRSFLLRNPNDKYEPFWEDEEK (SEQ ID NO: 8).

116. The chimeric protein of claim 111, wherein one or more of the cleavage sites is cleaved by a protease selected from the group consisting of factor XIa, factor Xlla, kallikrein, factor Vila, factor IXa, factor Xa, factor Ila (thrombin), Elastase-2, Granzyme- B, TEV, Enterokinase, Protease 3C, Sortase A, MMP-12, MMP-13, MMP-17, and MMP- 20.

117. The chimeric protein of claim 111 or 116, wherein one or more of the cleavage sites comprise an amino acid sequence selected from the group consisting of RRRR (SEQ ID NO: 9), RKRRKR (SEQ ID NO: 10), RRRRS (SEQ ID NO: 11), TQSFNDFTR (SEQ ID NO: 12), SVSQTSKLTR (SEQ ID NO: 13), DFLAEGGGVR (SEQ ID NO: 14), TTKIKPR (SEQ ID NO: 15), LVPRG (SEQ ID NO: 16), ALRPR (SEQ ID NO: 17), KLTRAET (SEQ ID NO: 18), DFTRVVG (SEQ ID NO: 19), TMTRIVGG (SEQ ID NO: 20), SPFRSTGG (SEQ ID NO: 21), LQVRIVGG (SEQ ID NO: 22), PLGRIVGG (SEQ ID NO: 23), IEGRTVGG (SEQ ID NO: 24), LTPRSLLV (SEQ ID NO: 25), LGPVSGVP (SEQ ID NO: 26), VAGDSLEE (SEQ ID NO: 27), GPAGLGGA (SEQ ID NO: 28), GPAGLRGA (SEQ ID NO: 29), APLGLRLR (SEQ ID NO: 30), PALPLVAQ (SEQ ID NO: 31), ENLYFQG (SEQ ID NO: 32), DDDKIVGG (SEQ ID NO: 33), LEVLFQGP (SEQ ID NO: 34 ), and LPKTGSES (SEQ ID NO: 35).

118. The chimeric protein of any one of claims 111 to 117, wherein the first cleavage site and the second cleavage site are identical or different.

119. The chimeric protein of any one of claims 1 to 118, which is polysialylated, pegylated, or hesylated.

120. A polynucleotide or a set of polynucleotides encoding the chimeric protein of any one of claims 1 to 120.

121. The polynucleotide of claim 120, further comprising a polynucleotide chain, which encodes PC5 or PC7.

122. A vector comprising the polynucleotide of claim 120 or 121 and one or more promoter operably linked to the polynucleotide or the set of polynucleotides.

123. The vector of claim 122, further comprising an additional vector, which comprises a polynucleotide chain encoding PC5 or PC7.

124. A host cell comprising the polynucleotide of any one of claims 120 or 121 or the vector of claim 122 or 123.

125. The host cell of claim 124, which is a mammalian cell.

126. The host cell of claim 125, wherein the mammalian cell is selected from the group consisting of HEK293 cell, CHO cell, and BHK cell.

127. A pharmaceutical composition comprising the chimeric protein of any one of claims 1 to 119, the polynucleotide of claim 120 or 121, the vector of claim 122 or 123, or the host cell of any one of claims 124 to 125, and a pharmaceutically acceptable carrier.

128. The composition of claim 127, wherein the FVIII protein has extended half-life compared to wild type FVIII protein.

129. The composition of claim 127 or 128, wherein the half-life of the FVIII protein is extended at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, or at least about 12 times longer than wild type FVIII.

130. The composition of claim 128 or 129, wherein the half-life of Factor VIII is at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, at least about 28 hours, at least about 29 hours, at least about 30 hours, at least about 31 hours, at least about 32 hours, at least about 33 hours, at least about 34 hours, at least about 35 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours.

131. The composition of any one of claims 127 to 130, which is administered by a route selected from the group consisting of topical administration, intraocular administration, parenteral administration, intrathecal administration, subdural administration and oral administration.

132. The composition of claim 131, wherein the parenteral administration is intravenous or subcutaneous administration.

133. The composition of any one of claims 127 to 132, which is used to treat a bleeding disease or condition in a subject in need thereof.

134. The composition of claim 133, wherein the bleeding disease or condition is selected from the group consisting of a bleeding coagulation disorder, hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, bleeding in the illiopsoas sheath and any combinations thereof.

135. The composition of claim 133 or 134, wherein the subject is scheduled to undergo a surgery.

136. The composition of any one of claims 133 to 135, wherein the treatment is prophylactic or on-demand.

137. A method of preventing or inhibiting binding of a FVIII protein with endogenous VWF comprising adding an effective amount of the chimeric protein of any one of claims 1 to 26 and 45 to 119, the polynucleotide of claim 120 or 121, the vector of claim 122 or 123, the host cell of any one of claims 124 to 126, or the composition of any one of claims 127 to 136 to a subject in need thereof, wherein the VWF fragment binds to the FVIII protein and thus prevents or inhibits binding of endogenous VWF.

138. A method of extending or increasing half-life of a FVIII protein, wherein the method comprises adding an effective amount of the chimeric protein of any one of claims 1 to 26 and 45 to 119, the polynucleotide of claim 120 or 121, the vector of claim 122 or 123, the host cell of any one of claims 124 to 126, or the composition of any one of claims 127 to 136 to a subject in need thereof, wherein the VWF fragment binds to the FVIII protein and thus extends or increases half-life of the FVIII protein.

139. A method of preventing or inhibiting clearance of a FVIII protein from a cell, wherein the method comprises adding an effective amount of the chimeric protein of any one of claims 1 to 26 and 45 to 119, the polynucleotide of claim 120 or 121, the vector of claim 122 or 123, the host cell of any one of claims 124 to 126, or the composition of any one of claims 127 to 136 to a cell comprising a FVIII protein or a polynucleotide encoding the FVIII protein, wherein the protein having VWF activity binds to the FVIII protein.

140. The method of any one of claims 137 to 139, wherein the subject is an animal.

141. The method of claim 140, wherein the animal is a human.

142. The method of claim 140, wherein the subject is suffering from hemophilia A.

143. A method of treating a bleeding disease or disorder in a subject in need thereof comprising administering an effective amount of the chimeric protein of any one of claims 1 to 26 and 45 to 119, the polynucleotide of claim 120 or 121, the vector of claim 122 or 123, the host cell of any one of claims 124 to 126, or the composition of any one of claims 127 to 136, wherein the bleeding disease or disorder is selected from the group consisting of a bleeding coagulation disorder, hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, and bleeding in the illiopsoas sheath.

144. The method of claim 143, wherein the treatment is prophylactic or on-demand.

145. The method of claim 143 or 144, wherein the effective amount is 0.1 μg/kg to 500 mg/kg.

146. The method of any one of claims 143 to 145, wherein the chimeric protein of any one of claims 1 to 119, the polynucleotide of claim 120 or 121, the vector of claim 122 or 123, the host cell of any one of claims 124 to 126, or the composition of any one of claims 127 to 136 is administered by a route selected from the group consisting of topical administration, intraocular administration, parenteral administration, intrathecal administration, subdural administration and oral administration.

147. The method of claim 146, wherein the parenteral administration is selected from the group consisting of intravenous administration, subcutaneous administration, intramuscular administration, and intradermal administration,

148. A method of making a chimeric protein, comprising transfecting one or more host cell with the polynucleotide of claim 120 or 121 or the vector of claim 122 or 123 and expressing the chimeric protein in the host cell.

149. The chimeric protein of any one of claims 27-119, wherein the XTEN insertion site is immediately downstream of residue 745 corresponding to the mature FVIII protein (SEQ ID NO: 4).

150. The chimeric protein of any one of claims 27-119, where the XTEN insertion sites are immediately downstream of residue 1656 and residue 1900 of the FVIII protein.

151. The chimeric protein of any one of claims 27-119, wherein the XTEN insertion sites are immediately downstream of residues 26, 1656, and 1900 of the FVIII protein.

152. The chimeric protein of any one of claims 27-119, wherein the XTEN insertion sites are immediately downstream of residues 403 and 745 of the FVIII protein.

153. The chimeric protein of any one of claims 27-119, wherein the XTEN insertion sites are immediately downstream of residues 745 and 1900 of the FVIII protein.

154. The chimeric protein of any one of claims 27-119, wherein the XTEN insertion sites are immediately downstream of residues 18 and 745 of the FVIII protein.

155. The chimeric protein of any one of claims 149-154, wherein the FVIII protein is a dual chain FVIII isoform.

156. The chimeric protein of any one of claims 149-154, wherein the FVIII protein is a single chain FVIII isoform.

157. The chimeric protein of any one of claims 149-156, comprising one XTEN.

158. The chimeric protein of any one of claims 149-156, comprising two XTENs.

159. The chimeric protein of any one of claims 149-156, comprising three XTENS are inserted.

160. The chimeric protein of any one of claims 149-157, wherein the XTEN is SEQ ID NO: 39 (AE288).

161. The chimeric protein of any one of claims 149-156 and 158, wherein the XTENs are SEQ ID NOs: 38 and 37 (AG144 and AE144).

162. The chimeric protein of any one of claims 149-156, and 159 wherein the XTENs are SEQ ID NOs: 37, 38 and 37 (AE144, AG144, and AE144).

163. The chimeric protein of any one of claims 149-156 and 157, wherein the XTENs are SEQ ID NOs: 38 and 39(AE144 and AE288).

164. The host cell of claim 124, wherein the PC5 or PC7 cleaves the D1D2 domains of VWF.

165. The chimeric protein of any one of claims 1 to 27, which comprises at least two XTENs, at least three XTENs, at least four XTENs, at least five XTENs, or at least six XTENs.